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. Marsh (Ed.)

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Membrane

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285

Current Topics
in Microbiology
and Immunology

Editors

R.W. Compans, Atlanta/Georgia

M.D. Cooper, Birmingham/Alabama

T. Honjo, Kyoto • H. Koprowski, Philadelphia/Pennsylvania

R Melchers, Basel • M.B.A. Oldstone, La Jolla/California

S. Olsnes, Oslo • M. Potter, Bethesda/Maryland

P.K. Vogt, La Jolla/California • H. Wagner, Munich

M. Marsh (Ed.)

Membrane
Trafficking
in Viral
Replication

With 19 Figures

4y Spri

ringer

Dr. Mark Marsh

Cell Biology Unit, MRC-LMCB

University College London

Gower Street

London, WC1E 6BT

United Kingdom

e-mail: m.marsh@ucl.ac.uk

Cover illustration: Lentivirus assembly. The principal image is an electron micrograph
of simian immunodeficiency virus (SIV) budding from the surface of an infected T cell
line. Budding figures, as well as immature and mature particles can be seen. The inset
shows a fluorescence micrograph of a human immunodeficiency virus (HIV) infected
macrophage stained to identify the viral gag protein. In these cells, virus assembly
occurs intracellularly in late endosomes (see p 219). Micrographs were provided by
Dr. Annegret Pelchen-Matthews, Cell Biology Unit, MRC-Lab oratory for Molecular Cell
Biology, University College London.

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Preface

Viruses are major pathogens in humans, and in the organisms with which
we share this planet. The massive health and economic burden these
agents impose has spurred a huge research effort to understand their most
intimate details. One outcome of this effort has been the production, in
many but certainly not all cases, of effective vaccines and therapies. An-
other consequence has been the realization that we can exploit viruses and
put them to work on our behalf. Viruses are still seen to have the most po-
tential as vehicles for gene delivery and other therapeutic applications.
However, their ability to exploit cellular functions to their own ends makes
viruses not only highly effective pathogens but also exquisite experimental
tools. Work with viruses underpins much of our current understanding of
molecular cell biology and related fields. For membrane traffic in particu-
lar, viruses have been crucial in providing insights into key cellular func-
tions and the molecular mechanisms underlying these events.

Viruses can be regarded as enveloped or non-enveloped agents. In en-
veloped viruses, a membrane derived from a membrane-bound compart-
ment of the host cell protects the genetic material. By contrast to enveloped
viruses, non-enveloped viruses protect their nucleic acid with a protein
shell assembled from polypeptides encoded in the viral genome. The fact
that enveloped viruses contain a membrane has made these agents particu-
larly useful for studies of membrane traffic. Some of the genetically less
complex viruses encode just one or two membrane proteins and have the
remarkable ability to take over a cell's protein synthetic capacity so that
within a few hours of being infected, a cell is dedicated to making only one
or two membrane proteins. These properties have made viruses such as
the rhabdovirus vesicular stomatitis virus (VSV) and the alphaviruses
Semliki Forest virus (SFV) and Sindbis virus extraordinarily useful in early
studies of the exocytic pathway. Indeed, the VSV G protein has been used
extensively in biochemical and morphological experiments, which have
led to key insights into the molecular details of this pathway.

Similarly, analysis of the mechanisms through which these, and other
viruses such as the orthomyxovirus influenza virus, generate new virions
has led to insights into glycoprotein synthesis and glycosylation, protein
folding, endoplasmic reticulum quality control, cell polarity, protein sort-
ing, etc. But these agents have proved to be of use not only in biosynthetic
events but in a variety of other processes involving cellular membrane sys-

Preface

terns. Studying virus entry led to many key insights into clathrin-depen-
dent receptor-mediated endocytosis. Now, nearly 25 years after those ini-
tial studies, similar experiments with different viruses are revealing aspects
of non-clathrin-mediated endocytosis. Similarly, viruses have provided
tractable experimental systems to analyse transport to the nucleus, mem-
brane fusion and other membrane transport events. Currently, the assem-
bly of some enveloped viruses is providing key insights into the function
of ESCRT proteins, the formation of multivesicular bodies and the sorting
of proteins for degradation in lysosomes.

It is this wealth of applications and the resulting knowledge that has
ensued from applying viruses to specific experimental problems that
has led to the present volume of Current Topics in Microbiology and Immu-
nology. This volume deals with a specific aspect of viral interactions with
cellular membrane systems and includes chapters on viral entry, viral
membrane fusion, viral membrane protein synthesis and transport, viral
replication, viral interaction with cytoskeletal systems and the nucleus, the
trafficking of viral membrane proteins and viral perturbation of host cell
protein trafficking. These chapters should provide new readers to the field
with a broad overview of the cellular membrane trafficking mechanisms
and viral interactions with these systems. They should also provide experts
in this field with reviews of the current state of each of the fields. Although
they incorporate a variety of topics, there are unfortunately areas that are
not covered in detail. This perhaps underlines the fact that this dynamic
and important field warrants frequent revisits.

It is no coincidence that a number of the contributors to the volume
have at one time or another worked with Ari Helenius. The composition of
the volume, at least in part, reflects the considerable contribution he has
made to these fields of study. I thank all the authors for their timely contri-
butions. I also thank Victoria, Megan, Kathleen and Molly for their sup-
port and patience.

Mark Marsh

List of Contents

Viral Entry

S.B. Sieczkarski and G.R. Whittaker 1

The Many Mechanisms of Viral Membrane Fusion Proteins

L.J. Earp, S.E. Delos, H.E. Park, and J.M. White 25

The Role of the Cytoskeleton During Viral Infection

K. Dohner and B. Sodeik 67

Nuclear Import in Viral Infections

U.F. Greber and M. Fornerod ,

109

Viral RNA Replication in Association with Cellular Membranes

A. Salonen, T. Ahola, and L. Kaariainen 139

Synthesis and Quality Control of Viral Membrane Proteins

C. Maggioni and I. Braakman 175

Receptor Modulation in Viral replication:

HIV, HSV, HHV-8 and HPV: Same Goal, Different Techniques

to Interfere with MHC-I Antigen Presentation

V. Piguet 199

Trafficking of Viral Membrane Proteins

R. Byland and M. Marsh 219

Subject Index 255

List of Contributors

(Their addresses can be found at the beginning of their respective chapters.)

Ahola, T. 139

Maggioni, C. 175

Braakman, I. 175

Marsh, M. 219

Byland, R. 219

Park, H.E. 25

Delos, S.E. 25

Piguet, V. 199

Dohner, K. 67

Salonen, A. 139

Earp, L.J. 25

Sieczkarski, S.B. 1

Fornerod, M. 109

Sodeik, B. 67

Greber, U.F. 109

White, J.M. 25

Kaariainen, L. 139

Whittaker, G.R. 1

CTMI (2004) 285:1-23
© Springer- Verlag 2004

Viral Entry

S. B. Sieczkarski 1 ' 2 • G. R. Whittaker 1 (El)

1 Department of Microbiology and Immunology, Cornell University,
C4127 Veterinary Medical Center, Ithaca, NY 14853, USA
grw7@ Cornell, edu

2 Department of Microbiology, School of Medicine and Biomedical Sciences,
University of Buffalo SUNY, Buffalo, NY 14214, USA

1 Introduction

2 Virus Receptor Binding 4

2.1 Receptors and Coreceptors 4

2.2 Viruses with Multiple Receptors and Multiple Use

of Receptors by Different Viruses 6

2.3 Receptor-Mediated Regulation of Endocytosis 7

2.4 Receptors and Infection of Polarized Epithelia 7

3 Virus Internalization 8

3.1 Direct Entry Through the Plasma Membrane 8

3.2 Internalization of Viruses into Endocytic Compartments 9

3.2.1 Clathrin and Non-Clathrin-, Non-Caveolae-Mediated Endocytosis. ... 9

3.2.2 Caveolae 11

3.2.3 Macropinocytosis 12

4 Postinternalization Trafficking 13

4.1 Rab Proteins and Endocytosis 13

4.2 Kinase-Mediated Regulation of Endocytosis 13

5 Membrane Penetration

6 Virus Uncoating . . .

7 Virus Entry as a Target for Antiviral Drugs 16

8 Perspectives

References

Abstract Virus entry is initiated by recognition by receptors present on the surface
of host cells. Receptors can be major mediators of virus tropism, and in many cases
receptor interactions occur in an apparently programmed series of events utilizing
multiple receptors. After receptor interaction, both enveloped and nonenveloped
viruses must deliver their genome across either the endosomal or plasma membrane
for infection to proceed. Genome delivery occurs either by membrane fusion (in the
case of enveloped viruses) or by pore formation or other means of permeabilizing

S.B. Sieczkarski • G.R. Whittaker

the lipid bilayer (in the case of nonenveloped viruses). For those viruses that enter
cells via endosomes, specific receptor interactions (and the signaling events that en-
sue) may control the particular route of endocytosis and/or the ultimate destination
of the incoming virus particles. Our conception of virus entry is increasingly becom-
ing more complex; however, the specificity involved in entry processes, once ascer-
tained, may ultimately lead to the production of effective antiviral agents.

Introduction

As obligate intracellular parasites, all viruses must have ways of entering
target cells to initiate replication and infection. The first step in virus en-
try is the recognition of the host cells through cell surface receptors.
This initial engagement can both mediate attachment and act as a prim-
er for subsequent conformational alterations in the virus. In many cases,
receptor interactions are important for defining the tropism of a virus
for a particular organism, tissue, or cell type. One important point to
note is that receptors not only serve as attachment points for viruses but
can induce conformational alterations in the bound ligand or induce sig-
naling events, which are a prerequisite for subsequent uncoating, traf-
ficking, or fusion events needed for infectious entry. After cell surface
attachment, animal viruses can become internalized in two principal
ways: by a direct mechanism at the cell surface (plasma membrane) or
after their internalization into cellular compartments, for example, en-
dosomes (Fig. 1). In the case of endocytic entry, internalization itself is
generally not sufficient for productive infection, as incoming viruses are
still part of the extracellular space while in endosomes. Therefore, endo-
cytosed viruses must penetrate the endosomal membrane to be released
into the cytoplasm. The endocytic pathway may also be used by viruses
that require a specific localization within the cell for successful infec-
tion.

This review is intended as a survey of the different routes of entry
used by animal viruses and will draw on both studies of the membrane
trafficking events required for entry and recent publications. For more
detail on entry of other viruses, such as bacteriophages, the reader is re-
ferred to the excellent recent review of Bamford and colleagues, which
describes common features of virus entry across the three domains of
life (Poranen et al. 2002). For more details on the entry mechanisms of
individual viruses, many classic reviews are available (Marsh and Hele-
nius 1989; Marsh and Pelchen-Matthews 1994, 2000; Young 2001).

Viral Entry

Internalization by endocytosis

Entry at the PM

MACROPIMOCYTOSIS

NON-CLATHflIN

NON-CAVEOLAE

ENDOCYTOSIS

CLATHRIN-
MEDIATED
ENDOCYTOSI!

CAVEOLAE

Fig. 1. Routes of virus internalization into host cells. Enveloped viruses may enter
host cells by direct fusion at the plasma membrane, depositing the viral nucleocap-
sid directly in the cytoplasm. Both enveloped and nonenveloped viruses may be in-
ternalized into host cells via endocytosis. Four primary endocytic routes have been
described, each known to be involved in virus entry. Clathrin-mediated endocyto-
sis can result in viral trafficking either through endosomes toward the lysosome or
into the recycling pathway. Non-clathrin-, non-caveolae-mediated endocytosis ap-
pears to traffic ligands toward the lysosome-targeted pathway. Viruses entering by
caveolae are usually targeted to the ER and possibly the Golgi. Macropinosomes
are capable of fusing with other vesicles of the endocytic pathways, such as early
endosomes

S.B. Sieczkarski • G.R. Whittaker

Virus Receptor Binding
2.1

Receptors and Coreceptors

The very first interaction of a virus with its host cell occurs via cell sur-
face receptors. This initiates a chain of events that is responsible for the
breach of the cell membrane. There is a wealth of information on cellular
receptors for viruses, which we can only summarize here. For more in-
formation, the reader is referred to Wimmer's monograph (Wimmer
1994) as well as several recent general reviews (Baranowski et al. 2001;
Mettenleiter 2002; Schneider- Schaulies 2000). For a detailed understand-
ing of regulation of receptor expression, see the chapter by Piguet in this
volume. In this article we focus on recent findings on virus receptors,
especially how they relate to the cell biology of virus entry, membrane
trafficking, and infection.

Virus-receptor interactions can be conveniently broken down into de-
fined areas. First, there is the basic concept that viruses interact with
cells initially by long-range, possibly nonspecific, electrostatic interac-
tions — based on attraction of the negatively charged cell surface with
positive charges on virus particles. A classic example of this is cell sur-
face heparan sulfate, a highly sulfated polysaccharide present in large
quantities on the surface of cells and in the extracellular matrix. Numer-
ous viruses, including many herpesviruses, papillomaviruses, para-
myxoviruses and dengue virus, use cell surface heparan sulfate to infect
a range of target cells (see Liu and Thorp 2002 for a review).

The view of heparan sulfate as a nonspecific receptor may, however,
turn out to be an oversimplification — a view exemplified by the her-
pesviruses. It was originally thought that interaction of cell surface hep-
aran sulfate with herpes simplex virus 1 (HSV-1), via glycoprotein C
(gC), was simply the preface to more specific protein-protein interac-
tions with other glycoproteins and coreceptors (see Campadelli-Fiume
et al. 2000; Mettenleiter 2002 for reviews). Such specific coreceptors in-
clude TNF-R, Prr2, nectinl, and PVR, which bind selectively to different
animal herpesviruses, via gD. However, in the case of HSV-1, unique
monosaccharide sequences (e.g., 3-O-sulfate) present on the cell surface
indicate that heparan sulfate could serve as a specific receptor, also via
gD, thus initiating virus entry (Shukla et al. 1999). Once specific cell sur-
face interactions have occurred, it is hypothesized that other herpesvirus
glycoprotein-cell surface interactions, possibly including gH and/or gB,

Viral Entry

then activate the fusion machinery between the virion envelope and the
plasma membrane, leading to translocation of the nucleocapsid into the
cell (see Campadelli-Fiume et al. 2000; Mettenleiter 2002 for reviews).

In addition to the herpesviruses, the most prominent example of a
dual receptor requirement occurs with human immunodeficiency virus
type-1 (HIV-1) binding. It has been known for many years that HIV-1
binds to cells of the immune system via cell surface CD4. However, this
interaction is not sufficient to mediate fusion. The virus needs to bind
so-called chemokine coreceptors, such as CXCR4 and CCR5, for produc-
tive infection (Berger et al. 1999; Overbaugh et al. 2001). Whereas CD4
usage is nearly universal, coreceptor usage varies between strains. The
interaction of gpl20 with the HIV-1 coreceptor is a now-classic example
of the induction of a conformational change in the viral glycoprotein
priming it for fusion. This coreceptor-induced conformational change
is in contrast to the simpler pH-mediated trigger used by viruses, such
as influenza, that bind a nonspecific receptor, in this case sialic acid
(Skehel and Wiley 2000).

HIV-1 also provides an example demonstrating that virus-receptor
binding does not necessarily lead to entry or productive infection of the
initially contacted cell, yet can profoundly influence viral pathogenesis.
In the case of HIV-1, the virus can bind via a lectinlike interaction to cir-
culating peripheral dendritic cells (DCs), via molecules such as DC-
SIGN (Geijtenbeek et al. 2000). Virus binding retains HIV-1 in an active
form on the cell surface while the DC undergoes maturation and migra-
tion to the regional lymph nodes. The outcome of this interaction is the
delivery of the virus to permissive T cells and initiation of infection
within the host.

One emerging theme of receptor-coreceptor interactions seems to be
that the complex series of conformational changes that follow binding of
pH-independent viruses, such as retroviruses, are not shared by pH-de-
pendent viruses, such as influenza virus, for which priming of fusion is
a much simpler and more rapid event, without extensive coreceptor in-
volvement. It remains to be seen how this might impact on a virus such
as avian leukosis virus (a retrovirus), which seems to be both pH- and
coreceptor dependent (Mothes et al. 2000). Considering that fusion is
being increasingly targeted as a point for antiviral intervention (see
Sect. 7), the speed and complexity of this interaction seem to have pro-
found implications for the ability of antiviral compounds to function.

S.B. Sieczkarski • G.R. Whittaker

2.2

Viruses with Multiple Receptors and Multiple Use
of Receptors by Different Viruses

Whether or not a virus uses a single protein or other moiety, or whether
a combination of receptor and coreceptor is used, the established para-
digm is of a simple and unique virus-receptor pairing. This has recently
been challenged in several systems, demonstrating that the same virus
can use multiple (individual) receptors, and that very different viruses
use identical receptors. For instance, as discussed above, it has become
apparent that herpesviruses, for example, HSV-1, can share the same re-
ceptor yet have a choice of coreceptors. Another important example in
which multiple (individual) receptors are used is measles virus (see Old-
stone et al. 2002; Yanagi et al. 2002 for reviews). The human CD46 was
originally identified as a ubiquitous cellular receptor for the Edmonston
and Halle strains of measles virus; however, this finding was challenged
as not all cell types seemed to be permissive, using CD46 as a receptor.
Subsequently SLAM (CD150) was identified as a principal receptor for
measles virus (and morbilliviruses in general), possibly accounting for
most cell types involved in measles virus pathology and pathogenesis.
The weight of evidence suggests that multiple (different) receptors are
important for measles virus, with the possibility that additional recep-
tors remain to be found.

Although in most cases individual viruses have their own distinct re-
ceptors, in some cases there can be competition for the same receptor
by quite different viruses. Perhaps the best studied example of this is the
coxsackie-adenovirus receptor or CAR (Bergelson et al. 1997). CAR is a
member of the immunoglobulin superfamily and mediates both attach-
ment and entry of these two viruses. CAR is sufficient for coxsackievirus
entry; however, for adenovirus (serogroup C), an additional cooperative
event is required after CAR binding — the interaction of the penton base
protein with an internalization receptor, the a v (3 3 and a Y j3 5 integrins (see
Nemerow 2000 for a review).

Another example of competition for the same receptor by different
viruses is illustrated by the poliovirus receptor, or PVR (CD 155), also a
member of the immunoglobulin superfamily. As well as acting as a re-
ceptor for poliovirus (Racaniello 1996), it is a coreceptor for two her-
pesviruses, bovine herpesvirus 1 and pseudorabies virus (Mettenleiter
2002). These viruses, along with poliovirus, all have neurotropism but it
remains to be seen whether the specific use of PVR is an important fac-
tor in pathogenesis. In the case of poliovirus it is clear that species infec-

Viral Entry

tivity, but not necessarily tissue tropism, is determined by the receptor
(Nomoto et al. 1994).

2.3

Receptor-Mediated Regulation of Endocytosis

One feature of virus-receptor interaction is that the virus may be physi-
cally linked to a cellular receptor, which has inherent information for en-
docytic traffic. This allows the virus to utilize endocytic trafficking sig-
nals inherent in the receptor. Whereas in most cases binding of cellular
ligands induces signaling events responsible for receptor clustering and
internalization into defined endocytic pathways, viruses are inherently
multivalent and may be able to mimic ligand-induced receptor internal-
ization in the absence of normal signals. One well-studied example is the
transferrin receptor, which undergoes clathrin-mediated internalization
and enters the recycling pathway of endocytosis. Viruses such as canine
parvovirus bind and enter cells via the transferrin receptor (Parker et al.
2001; Hueffer et al. 2003) and appear to enter a recycling endocytic com-
partment before genome delivery, possibly via late endosomes, to the cy-
tosol (Suikkanen et al. 2002). Other viruses, such as mouse mammary
tumor virus (a retrovirus), that also use transferrin receptor for entry
may direct their endocytic uptake in a similar manner (Ross et al. 2002).
Such receptor-defined trafficking may not be limited to the transferrin
receptor, as other well-characterized, clathrin-dependent receptors, such
as LDL receptor-related proteins, are used by rhinoviruses (Hofer et al.
1994). It is presently unclear whether the recent finding that influenza
virus may have alternative internalization routes, via clathrin- or non-
clathrin-mediated endocytosis (Sieczkarski and Whittaker 2002), is ac-
counted for by the nonspecific receptor (sialic acid) used by the virus.

2.4

Receptors and Infection of Polarized Epithelia

Many viruses infect polarized epithelia, and it has been appreciated for
some time that expression of receptor molecules has special significance
for viruses that infect epithelial cells (Tucker et al. 1994). One recently
appreciated finding is that cell adhesion molecules are used as entry re-
ceptors by several viruses, including reovirus (junctional adhesion mol-
ecule, JAM), herpes simplex virus (nectinl and 2), and CAR (see Spear
2002 for a review). As such, cell adhesion molecules localize to junction-
al complexes and account for the integrity of epithelia; they can have

S.B. Sieczkarski • G.R. Whittaker

pronounced effects on virus spread within the organism. The use of cell
adhesion molecules might be considered unusual in that the receptor is
not typically accessible from the apical face of the epithelium, where the
virus enters its host. In this case, successful infection might depend on
damage to the epithelium. In addition, spread of virus might be compro-
mised without breakdown of the epithelium. Recent studies on adenovi-
rus show that the fiber-knob protein of adenovirus disrupts interactions
between the CAR receptors of adjacent cells and allows virus to exit
across the airway epithelium (Walters et al. 2002). After infection, hu-
man airway epithelia first release adenovirus to the basolateral surface.
Virus then travels between epithelial cells by disrupting CAR- CAR inter-
actions, to emerge on the apical surface for viral spread. Thus internal-
ization with adhesion molecules can direct the spread of virus infection
within the host and has profound implications for the design of gene
therapy vectors.

Virus Internalization

3.1

Direct Entry Through the Plasma Membrane

Perhaps the simplest route of entry into the cell is directly through the
plasma membrane. Indeed, this route of entry is clearly used by a range
of different viruses. Those viruses generally considered to enter through
the plasma membrane include paramyxovirus and herpesvirus (Lamb
1993; Spear 1993). Despite the apparent simplicity, there are some dis-
advantages to this route of entry. First, the cell contains a barrier of cor-
tical actin through which the virus must navigate (see Marsh and Bron
1997 and the chapter by Dohner and Sodeik, this volume, for a discus-
sion). Second, the endosome often carries out the critical function of
acidification, needed for triggering of fusion (see the chapter by Earp et
al., this volume). By default, therefore, viruses that enter through the
plasma membrane must be pH independent. Third, the endosome may
also provide a specific chemical environment, such as ion concentration
or redox state needed to allow penetration. Finally, endocytic traffic
may deliver the genome deep into the interior of the cell, close to the
nucleus.

Recently, retroviruses have been actively studied with regard to direct
fusion vs. endocytosis. Although classically defined as being pH indepen-

Viral Entry

dent, some retroviruses are now thought to require pH for fusion (see
the chapter by Earp et al, this volume) and presumably enter via endocy-
tosis. Of particular note is the finding that the avian leukosis virus, a ret-
rovirus, shows a substantial inhibition of infection in cells expressing
dominant-negative dynamin, a cellular molecule essential for endocytosis
(Mothes et al. 2000). However, another retrovirus, Moloney murine leu-
kemia virus, showed no effects on expression of dominant-negative dy-
namin (Lee et al. 1999). It is unclear whether these apparently contradic-
tory data for retroviruses represent differences in the individual viruses
or in the assay conditions used, or whether dynamin is required for other
membrane traffic events important for virus assembly.

HIV is another retrovirus that has been actively studied in this re-
gard. Whereas the majority of virus does get endocytosed (especially
under high-MOI conditions), this pool of virus does not seem to enter
the cytosol and is destroyed in lysosomes (Fredericksen et al. 2002). Un-
der certain circumstances this endocytosed virus may be infectious
(Fackler and Peterlin 2000); however, the major route of HIV entry is di-
rectly through the plasma membrane.

3.2

Internalization of Viruses into Endocytic Compartments

Endocytosis has emerged as the principal route of entry into host cells
for the majority of virus families (Russell and Marsh 2001; Sieczkarski
and Whittaker 2002). In principle, such viruses have several choices for
internalization, based on the available trafficking pathways in the cell
(see Fig. 1). These can be broken down into four distinct pathways: (1)
clathrin, (2) non-clathrin, non-caveolae pathways, (3) caveolae, and (4)
macropinocytosis. However, individual viruses within a family, or the
same virus in different cell types, may use different internalization
routes.

3.2.1

Clathrin and Non-Clathrin-, Non-Caveolae-Mediated Endocytosis

As befits its major role in endocytosis, clathrin has been shown to play a
major role in the internalization of many viruses. Traditionally, several
viruses, including influenza virus, VSV, and SFV, were identified in
clathrin-coated vesicles at early times of internalization (e.g., 5 min)
based on the presence of an electron-dense coat by transmission elec-
tron microscopy (Marsh and Helenius 1980; Matlin et al. 1981, 1982).

S.B. Sieczkarski • G.R. Whittaker

However, noncoated vesicles were also observed in these experiments,
due either to the release of clathrin or the presence of non-clathrin-me-
diated pathways. Subsequently, a role for clathrin in the case of SFV has
been shown by microinjection of anti-clathrin antibodies (Doxsey et al.
1987). The expression of dominant-negative Epsl5 (which arrests clath-
rin-coated pit assembly at the cell surface) has more recently confirmed
a role for clathrin in SFV and VSV entry (Sieczkarski and Whittaker
2002; V. Yau and G. Whittaker, unpublished results). Other viruses in
which clathrin is required for entry are adenovirus (Meier et al. 2002),
parvovirus (Parker and Parrish 2000), Hantaan virus (Jin et al. 2002),
and Sindbis virus (Carbone et al. 1997). In the case of influenza virus,
the situation is much less clear — in that elimination of clathrin function
showed no inhibition of virus infection or replication (Sieczkarski and
Whittaker 2002). Thus influenza may have the ability to use either clath-
rin or non-clathrin endocytic pathways for productive infection.

For many other virus families, the reliance on clathrin is also much
less clear-cut. One notable example is the Picornaviridae. Potassium de-
pletion has shown a role for clathrin in human rhinovirus infection
(Bayer et al. 2001; Madshus et al. 1987). In the case of rhinovirus, it may
be that different virus serotypes use different pathways. The role of acid-
ification during rhinovirus entry is also uncertain (Huber et al. 2001).
For other family members, such as poliovirus, the data are equally un-
clear. Poliovirus has been proposed to enter independently of clathrin
(DeTulleo and Kirchausen 1998), and a pH-independent route of entry
seems likely (Perez and Carrasco 1993). However, it is still unclear
whether endosomes are required for entry, or whether penetration can
occur directly through the plasma membrane (for discussion, see Hogle
2002). Poliovirus illustrates two important points that must be remem-
bered when thinking about virus entry. First, the use of an endocytic
route is not necessarily tied to pH dependence, and second, a pH-inde-
pendent virus may choose to enter through the cell surface or through
endosomes, depending on specific circumstances.

Enteroviruses, on the other hand, appear to use non-clathrin path-
ways that are dependent on cell surface lipid rafts (Stuart et al. 2002). In
this case, the use of a novel pathway seems to be directly related to the
receptor used by the virus (decay accelerating factor, or DAF). One new-
ly-added group in the Picornaviridae are the echoviruses. Human pare-
chovirus-2 was shown to enter cells via clathrin-dependent endocytosis
but to traffic to the ER (Joki-Korpela et al. 2001). Thus these echoviruses
may show features similar to simian virus 40 (SV40). Echovirus-1 shows

Viral Entry

no colocalization with markers of the clathrin pathway but appears to
use caveolae for entry (Marjomaki et al. 2002); see Sect. 3.2.2.

Another virus family that makes use of both clathrin and non-clath-
rin pathways for entry is the Polyomaviridae. SV40 is well established to
use caveolae as a route of entry (Kartenbeck et al. 1989; Pelkmans et al.
2001; see Sect. 3.2.2). However, one recent study reports incoming mu-
rine polyomavirus having no colocalization with caveolin and being in-
dependent of both clathrin and dynamin (Gilbert and Benjamin 2000).
In glial cells, however, the human polyomavirus JC virus enters by pH-
sensitive, clathrin-dependent endocytosis, in contrast to the pH-inde-
pendent, caveolar pathway used by SV40 in the same cell (Ashok and At-
wood 2003; Pho et al. 2000). Clearly, more investigation is required to
clarify the role of clathrin and non-clathrin pathways in the entry of the
different members of the Polyomavirinae.

Although generally considered to use direct fusion at the plasma
membrane, herpesviruses can also use endocytosis. A role for clathrin
has been implicated in the uptake of the Epstein-Barr virus (EBV) into B
cells of the immune system, but not in epithelial cells (Miller and Hutt-
Fletcher 1992). Other evidence for herpesviruses utilizing the endocytic
pathway is currently emerging, with HSV-1 also believed to utilize pH-
dependent endocytosis to enter certain cell types (Nicola et al. 2003).

3.2.2

Caveolae

Caveolae have emerged as a somewhat specialized route of virus entry,
notably for SV40. The uptake of SV40 is somewhat unusual, in that in-
coming virions accumulate in the smooth ER (Kartenbeck et al. 1989).
Entry via caveolae was recently shown by virus colocalization with en-
dogenous caveolin, a major component of caveolae (Norkin 1999), and
more specifically with the use of GFP- tagged caveolin- 1 combined with
video microscopy — which showed the delivery of SV40 from caveolae
directly to the ER, utilizing dynamin and local actin recruitment but by-
passing the traditional endosome/lysosome system (Pelkmans et al.
2001, 2002). However, it is also possible that virus moves transiently
through the Golgi en route to the ER (Norkin et al. 2002; Richards et al.
2002). Other notable features of SV40 entry via caveolae are that the ki-
netics are slow compared to the clathrin-coated vesicle pathway, that the
vesicles do not become acidified, and that internalization is an active
process driven by virus binding.

S.B. Sieczkarski • G.R. Whittaker

Colocalization with endogenous caveolin has also implicated caveolae
in the entry of mouse polyoma virus (Richterova et al. 2001); however,
incoming murine polyomavirus was shown not to colocalize with cave-
olin (Gilbert and Benjamin 2000). Despite extensive investigation, it ap-
pears that the caveolar route of virus entry may be a relatively special-
ized event, limited to SV40 as well as other viruses that are routed to the
ER/Golgi during entry, such as echoviruses (Marjomaki et al. 2002),
although caveolae may not be obligatory in the latter case (Joki-Korpela
et al. 2001; see Sect. 3.2.1).

3.2.3

Macropinocytosis

Macropinocytosis is generally considered to be a nonspecific mechanism
for internalization, in that it is not reliant on ligand binding to a specific
receptor. Instead, formation of endocytic vesicles occurs as a response
to cell stimulation, particularly at sites of membrane ruffling. The find-
ing that macropinosomes have the ability to become acidified and can
intersect with endocytic vesicles makes them possible routes of entry for
a wide variety of viruses; however, detailed mechanisms have not
emerged in most cases. Experiments have recently been used to demon-
strate macropinocytic uptake of HIV-1 into macrophages (Marechal et
al. 2001), although most viruses internalized by this route were probably
noninfectious. Although the reasons remain unclear, virus-induced en-
docytic activity is required for adenovirus type 2 penetration even
though the virions themselves are internalized in clathrin-coated vesicles
(Meier et al. 2002).

Macropinocytosis is known to be a major route of entry into antigen-
presenting cells like dendritic cells, and the ultimate importance of
macropinocytosis in viral infection may therefore turn out to be in the
presentation of the invading virus to the immune system, rather than in
its primary infection of epithelial surfaces.

Viral Entry

Postinternalization Trafficking

4.1

Rab Proteins and Endocytosis

One class of molecules that regulate membrane traffic events involved in
endocytosis is the Rab family of small GTPases (Somsel Rodman and
Wandinger-Ness 2000). The involvement of Rab GTPases in virus entry
is at a preliminary stage of investigation. Adenovirus uptake was in-
creased by overexpression of wild-type Rab5 (which controls entry into
the early endosome) and decreased by dominant-negative Rab5 (Rauma
et al. 1999). We recently carried out a more comprehensive study of vi-
rus entry, using influenza virus, SFV, and VSV. All of these viruses were
sensitive to Rab5 inhibition, but only influenza was affected by Rab7 in-
hibition (which controls formation of late endosomes and lysosomes)
(Sieczkarski and Whittaker 2003). These data suggest that influenza vi-
rus has a requirement for functional late endosomes during entry.

The above viruses seem to rapidly enter and escape from their respec-
tive endocytic vesicles (typically within 20-30 min). Although the role
of Rab proteins was not addressed, parvovirus is suggested to have a dif-
ferent type of endocytic interaction. Canine parvovirus enters a slow re-
cycling pathway, normally typified by the presence of Rab 11, and even-
tually breaks through to the late endosome for productive infection
(Parker and Parrish 2000; Suikkanen et al. 2002).

Rab proteins are also convenient markers for colocalization studies,
as they tend to be organelle specific in their localization. For example,
Miyazawa et al. (Miyazawa et al. 2001) used Rab7 to show the localiza-
tion of adenovirus subgroup B (Ad7) to late endosomes. In contrast,
these authors showed that the related (and more commonly studied) ad-
enovirus subgroup C does not enter the late endosome but seems to
penetrate from the early endosome. Thus very similar viruses may show
selective endocytic traffic, which could have profound implications for
their pathogenesis.

4.2

Kinase-Mediated Regulation of Endocytosis

As befits their role as major signaling pathways in cells, virus endocytic
events are regulated by phosphorylation. Attention has focused on two
kinase families, the PI 3-kinases (PI3K) and protein kinase C (PKC). For
adenovirus, the initial interaction of the virus with cell surface integrins

S.B. Sieczkarski • G.R. Whittaker

activates PI3K, which in turn affects virus endocytosis. Inhibition of
PI3K reduces adenovirus internalization to approximately 30% of con-
trol samples (Li et al. 1998). It has not yet been shown at which point in
entry adenovirus internalization is arrested by PI3K inhibition.

For many years, PKC has also been implicated in virus entry process-
es. The entry of several enveloped viruses, including rhabdoviruses, al-
phaviruses, poxviruses, and herpesviruses, has been proposed to require
PKC based on the action of protein kinase inhibitors such as H7 and
staurosporine (Constantinescu et al. 1991). More recently, it was shown
that the successful entry of adenovirus type 2 requires PKC. In the pres-
ence of calphostin C, an inhibitor of the classic and novel PKC isoforms,
adenovirus is prevented from escaping endosomes and accumulates in
cytoplasmic vesicles near the cell periphery. Bisindolylmaleimide I, a
broad-spectrum, highly specific PKC inhibitor, as well calphostin C, pre-
vents influenza virus entry and subsequent infection (Root et al. 2000; S.
Sieczkarski and G. Whittaker, unpublished results). Other viruses seem
to require PKC for infection, but not specifically for virus entry (S.
Sieczkarski and G. Whittaker, unpublished results). A specific role for
PKC in influenza virus entry is reinforced by recent data from our labo-
ratory in cells overexpressing a kinase-dead form of the PKQ3II isotype,
but not PKCa. In these cells, influenza virus entry was arrested at the
level of the late endosome without any apparent defect in endosome
acidification (Sieczkarski et al. 2003). It remains to be seen which specif-
ic isoforms of PKC are involved in the entry of different viruses, and at
which point in endocytic trafficking they act.

Membrane Penetration

To replicate, viruses must deliver their genomes across a cellular mem-
brane system to the cytoplasm. For enveloped viruses, this occurs by fu-
sion of the virion envelope with a cellular membrane. As virus fusion is
covered extensively in the chapter by Earp et al., this volume, it will not
be discussed here. Nonenveloped viruses deliver their genomes by a less
obvious means and breach the hydrophobic barrier of the membrane by
pore formation or other mechanism(s). In some cases this process is
somewhat understood, but for many nonenveloped viruses it is still a
big mystery.

Picornaviridae are the best-studied nonenveloped virus for penetra-
tion. In the case of poliovirus, many molecular events are now recog-

Viral Entry

nized — based on extensive structural studies of the molecules involved
(reviewed in Hogle 2002). After receptor interaction, it is generally
thought that conformational changes in the virus result in the formation
of a functional intermediate (the A particle), where the hydrophobic
N- terminus of VP1, and possibly a myristate group on VP4, possibly
combined with changes in Ca 2+ concentrations, allow membrane bind-
ing and pore formation. It has been proposed that there are common el-
ements for entry of both enveloped (such as the extensively character-
ized influenza HA) and nonenveloped viruses (such as poliovirus),
based on similar mechanisms for receptor binding leading to release
of the virus from a metastable state and exposure of hydrophobic se-
quences (Hogle 2002).

One distinguishing feature of membrane penetration for picor-
naviruses appears to be the presence of a small, defined pore, through
which the genome translocates. This localized event may be crucial, es-
pecially in the case of plasma membrane penetration, where a more gen-
eralized perforation or rupture would damage the host cell. Although
low pH per se may not be a key factor in penetration of picornaviruses,
combinations of concanamycin (a vacuolar H + -ATPase inhibitor) with
valinomycin (an ionophore that promotes K + efflux from cells) can pre-
vent poliovirus entry into cells (Iruzun and Carrasco 2001). Therefore, it
appears that the virus requires an intact K + gradient across the mem-
brane in order to uncoat and enter cells. In a similar manner, K + con-
centrations have been proposed to play a role in the exposure of a hydro-
phobic conformer of the ul protein, which mediates membrane disrup-
tion or perforation during entry of reoviruses (Chandran et al. 2002).

Another intriguing finding is that parvoviruses encode a sequence
with similarity to cellular phospholipases within their VP1 capsid pro-
tein unique region (Girod et al. 2002; Zadori et al. 2001). It is proposed
that conformation changes occurring within the acidic environment ex-
pose the phospholipase activity, which can mediate localized disruption
of the endosomal membrane and allow capsid entry into the cytosol.
This seems to be a specialized event for these viruses, and a similar
mechanism has not yet been described for other viruses.

Virus Uncoating

After membrane penetration, viruses must deliver their genome con-
tents to the site of replication. As this event is often tied to the processes

S.B. Sieczkarski • G.R. Whittaker

of cytoplasmic and nuclear transport, it will not be covered extensively
here, and the reader is referred to other chapters in this volume and to
previous reviews (Greber et al. 1994; Smyth and Martin 2002). However,
one virus family that does not make direct use of either the cytoplasmic
or nuclear transport machineries are reoviruses. These viruses are
somewhat unusual in that they require low pH during virus entry, not
for membrane penetration but for nucleocapsid uncoating (Sturzen-
becker et al. 1987). Reoviruses have a very stable double-shelled capsid,
which is disrupted by proteases during virus entry. The requirement for
low pH is generally not thought to be a direct effect, but rather low pH
is needed for activation of proteases, such as lysosomal cathepsin L and
B (Ebert et al. 2002), that cleave the outer capsid shell, allowing genome
release. In some cases, however, reoviruses can bypass the lysosomal re-
quirement and utilize exogenous proteases (Golden et al. 2002). It is be-
lieved that in vivo such proteases present in the lumen of the gut can
profoundly influence virus pathogenesis, which leads to the concept of
two modes of virus entry for reovirus — cell surface or lysosomal (Borsa
et al. 1979).

Virus Entry as a Target for Antiviral Drugs

As a target for therapeutic intervention, virus entry offers the advantage
that the number of incoming particles is often small, maximizing any in-
hibitory action of an antiviral drug. Indeed, the newest generation of
anti-HIV drugs target entry into host cells (O'Hara and Olson 2002).
HIV entry inhibitors fall into two main groups: those targeting fusion
(discussed in the chapter by Earp et al., this volume) and those designed
as coreceptor inhibitors. In the latter group, two small molecules, SCH-C
and AMD 3100 (specific for CCR5 and CXCR4, respectively), are cur-
rently in clinical trials, along with a humanized monoclonal antibody to
CCR5.

Other antiviral agents targeting virus entry include amantadine and
the WIN compounds. Amantadine was one of the first antiviral drugs to
be used in patients, and it targets the influenza A virus M2 ion channel
(Hay et al. 1985; Pinto et al. 1992). Amantadine and its derivative riman-
tadine are still in use today but suffer from problems due to the emer-
gence of resistant viruses. The WIN compounds target the "canyon"
present as part of the receptor-binding pocket of picornaviruses and
prevent virus uncoating (Lewis et al. 1998). Although powerful antivirals

Viral Entry

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S.B. Sieczkarski • G.R. Whittaker

in experimental systems, they have not been successful therapeutically,
because of the many possible serotypes of picornaviruses and variable
binding affinities of the drug with the hydrophobic pocket of the can-
yon. However, an advance on the WIN compounds has occurred with
the development of a new generation of capsid-function inhibitors, such
as Pleconaril (Rotbart 2002).

Perspectives

The classic notion of a virus binding to a single receptor to enter cells
through a single defined internalization mechanism is quickly being
overtaken by a more complex picture of virus entry. New findings, such
as specific coreceptor usage and virus attachment to multiple (different)
receptors (summarized in Table 1), have called into question our notions
of how other viruses function. For instance, viruses known to bind to a
nonspecific receptor may turn out to also have a more specific corecep-
tor. The use of multiple receptors may allow understanding of the patho-
genic differences among virus strains. Beyond the initial entry steps,
viruses also appear to follow more complex intracellular trafficking
routes than imagined previously. Through kinase cascades, viruses may
be able to alter cellular signaling pathways to control their endocytic
movement. The recognition of new endocytic entry pathways, such as
non-clathrin-, non-caveolae-mediated endocytosis and macropinocyto-
sis, has coincided with the discovery that many virus families appear to
use multiple routes of entry. The specifics of these entry decisions, be
they based on cell type, receptor availability, or extracellular conditions,
have yet to be determined. The complexity involved in virus entry may
make discovering treatments targeting this stage of the infectious cycle
more challenging, but the specificity involved in the processes, once as-
certained, may ultimately lead to the production of effective antiviral
agents.

Acknowledgements We thank Ruth Collins and Karsten Hiiffer for critical reading of
this manuscript. Work in the authors' laboratory is supported by the American Lung
Association and the National Institutes of Health.

Viral Entry

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CTMI (2004) 285:25-66
© Springer- Verlag 2004

The Many Mechanisms

of Viral Membrane Fusion Proteins

L. J. Earp 1 • S. E. Delos 2 • H. E. Park 1 • J. M. White 2 (^)

1 Department of Microbiology, University of Virginia, Charlottesville, VA, USA

2 Department of Cell Biology, School of Medicine, UVA Health System,
P.O. Box 800732, Charlottesville, VA 22908-0732, USA

jw7g@v irgin ia. edu

1 Introduction

2 Activation of Viral Fusion Proteins 27

2.1 Low pH Activation 28

2.2 Receptor Activation at Neutral pH 29

2.3 "Two-Step" Activation 29

3 Classification of Fusion Proteins Based on Structural Criteria 30

4 Examples of Fusion Activation Mechanisms 32

4.1 Influenza HA (Class I Fusion Protein, Low pH) 33

4.2 HIV Env (Class I Fusion Protein, Neutral pH) 34

4.3 Paramyxovirus F Proteins (Class I Fusion Protein, Neutral pH,
Attachment Protein Assisted) 36

4.4 TBE E and SFV El (Class II Fusion Proteins, Low pH) 38

5 Membrane Dynamics During Fusion 40

6 Membrane-Interacting Regions of Viral Fusion Proteins 41

6.1 The Fusion Peptide 42

6.1.1 Structure of N-terminal Fusion Peptides 43

6.1.2 Structure of Internal Fusion Peptides 44

6.1.3 Roles of Fusion Peptides 45

6.2 The Transmembrane Domain 45

6.3 The Juxtamembrane Region of the Ectodomain 46

6.4 The Cytoplasmic Tail 47

7 Rafts in Viral Membrane Fusion

8 Inhibitors of Viral Fusion 49

8.1 Inhibition of Helix Bundle Formation 50

8.2 Inhibition of Other Steps in Fusion 51

9 Perspectives

References

L.J. Earp et al.

Abstract Every enveloped virus fuses its membrane with a host cell membrane,
thereby releasing its genome into the cytoplasm and initiating the viral replication
cycle. In each case, one or a small set of viral surface transmembrane glycoproteins
mediates fusion. Viral fusion proteins vary in their mode of activation and in struc-
tural class. These features combine to yield many different fusion mechanisms. De-
spite their differences, common principles for how fusion proteins function are
emerging: In response to an activating trigger, the metastable fusion protein con-
verts to an extended, in some cases rodlike structure, which inserts into the target
membrane via its fusion peptide. A subsequent conformational change causes the fu-
sion protein to fold back upon itself, thereby bringing its fusion peptide and its
transmembrane domain — and their attached target and viral membranes — into inti-
mate contact. Fusion ensues as the initial lipid stalk progresses through local hemi-
fusion, and then opening and enlargement of a fusion pore. Here we review recent
advances in our understanding of how fusion proteins are activated, how fusion pro-
teins change conformation during fusion, and what is happening to the lipids during
fusion. We also briefly discuss the therapeutic potential of fusion inhibitors in treat-
ing viral infections.

Keywords Membrane fusion protein • Class I fusion protein • Class II fusion protein •
Influenza HA • HIV Env • Low-pH activation • Receptor activation • Conformational
changes • Membrane dynamics • Anti-fusion antivirals

Introduction

Fusion of enveloped viruses with host cells remains an important topic
of research for two major reasons. First, it has recently become clear that
fusion is a good target for therapeutic intervention (Kilby et al. 1998).
Second, viral fusion reactions continue to serve as models for cellular
fusion events. Although several viral fusion proteins, such as influenza
hemagglutinin (HA) and the human immunodeficiency virus (HIV) en-
velope glycoprotein (Env), have emerged as paradigms, it is important
to realize that there are many distinguishing features among viral fusion
proteins (Table 1). Viral fusion proteins can be activated for fusion by
different mechanisms. They have also been classified according to struc-
tural criteria. For some viruses, the viral receptor does not actively par-
ticipate in fusion, whereas for others, one or more receptors are essential
players. The location of the fusion peptide, critical for fusion, can vary.
Finally, whereas some viruses require a single viral glycoprotein to me-
diate fusion, others require multiple viral glycoproteins. There are many
excellent recent reviews on viral fusion and the glycoproteins that
mediate this process (Durell et al. 1997; Eckert and Kim 2001; Heinz and

The Many Mechanisms of Viral Membrane Fusion Proteins

Table 1. Viral membrane fusion

proteins

Family

Viral proteins

pHof

Class

Fusion peptide

needed

fusion

Orthomyxovirus

Low

N-terminal

Alphavirus

Low

Internal

Flavivirus

Low

Internal

Rhabdovirus

Low

•

Internal

Bunyavirus

G1/G2

Low

•

Arenavirus

Low

•

Filovirus

Low a

Internal

Retrovirus

Env

Neutral b

N-terminal, internal

Paramyxovirus

F,HN

Neutral

N-terminal

Herpesvirus

gB, gD, gH :

• gL

Neutral

•

Coronavirus

Neutral

Internal

Poxvirus

N.D.

Neutral

•

Hepadnavirus

Neutral? d

•

Iridovirus

N.D.

•

a Inferred from infectivity assays.

b Most retroviruses fuse at neutral pH. MMTV appears to require low pH [Ross et
al. (2002) PNAS 99:12386-90] to fuse. Avian retroviruses require receptor priming
at neutral pH followed by exposure to low pH [Mothes et al. (2000) Cell 103:679-89;
see text for a discussion of this model].

c Coronaviruses possess heptad repeats [Chambers et al. (1990) J Gen Virol
71:3075-80] characteristic of class I viral fusion proteins. Recent work indicates that
they are, indeed, class I fusion proteins [Bosch et al. (2003) J Virol 77:8801-11].

d With infectivity assays, hepadnavirus uptake was shown to be pH-independent
[Hagelstein et al. (1997) Virology 229:292-4]. However, recent studies have shown
that duck hepatitis B virus may require low pH [Grgacic et al. (2000) J Virol
74:5116-22].

e The S protein contains a stretch of amino acids predicted to be a fusion peptide
but has not been further characterized.

Allison 2001; Skehel and Wiley 2000; Weissenhorn et al. 1999). The goal
of this review is to give the reader an appreciation for the diversity of
viral fusion mechanisms.

Activation of Viral Fusion Proteins

All fusion proteins exist on virion surfaces in a metastable state in which
the fusion peptide, a critical hydrophobic sequence, is hidden or shield-
ed within the glycoprotein oligomer (Carr et al. 1997; Hernandez et al.

L.J. Earp et al.

1996; Rey et al. 1995; Skehel and Wiley 2000; Wilson et al. 1981). After
activation, the fusion peptides are rendered accessible for interaction
with a target membrane. A major distinction among viral fusion pro-
teins is the "trigger" for activation. There are two well-recognized mech-
anisms: (1) exposure to low pH and (2) specific interactions with target
cell receptors at neutral pH. A third mechanism involving receptor
priming at neutral pH followed by further activation at low pH was re-
cently proposed (Mothes et al. 2000).

2.1

Low pH Activation

Orthomyxoviruses, togaviruses, flaviviruses, rhabdoviruses, bunyavirus-
es, arenaviruses, and, apparently, filoviruses require low pH to fuse with
target membranes (Table 1) (Doms et al. 1985; Gaudin et al. 1999b; Steg-
mann et al. 1987; White and Helenius 1980). These viruses are endocyto-
sed after binding to the target cell surface. The low-pH environment of
the endosome activates the viral fusion protein to convert from a meta-
stable state to one that is capable of driving fusion. Although the pres-
ence of a receptor may modulate the rate or extent of fusion (Ohuchi et
al. 2002; Stegmann et al. 1996; White et al. 1982), receptors are not es-
sential for low-pH-dependent fusion. Low-pH-dependent fusion general-
ly occurs within seconds to minutes at 37°C but can also occur, albeit
more slowly, at T<22°C.

Four main techniques have been used to assess whether a virus re-
quires low pH to fuse. The first technique is testing the effects of agents,
such as bafilomycin, that inhibit endosomal acidification. In some stud-
ies of this type, fusion has been measured directly by assessing the
transfer of fluorescent probes from the virus to the target cell (Earp et
al. 2003; Irurzun et al. 1997; Zarkik et al. 1997). In others, fusion has
been inferred by monitoring postfusion events, such as the synthesis of
viral DNA (Mothes et al. 2000).

A second test is to assess whether fusion of bound virions can be in-
duced by briefly warming virus-cell complexes in low-pH medium
(Mothes et al. 2000; White et al. 1980). A third test is to assess whether
pretreatment of virions at low pH (in the absence of target membranes)
inactivates the virus for fusion. Some (Bron et al. 1993; Corver et al.
2000; Di Simone and Buchmeier 1995; Korte et al. 1999; Nir et al. 1990;
Stegmann et al. 1987), but not all (Puri et al. 1988), viruses that fuse at
low pH can be inactivated by this method. Viral fusion proteins that are
inactivated by low pH undergo irreversible conformational changes. In

The Many Mechanisms of Viral Membrane Fusion Proteins 29

the case of X:31 HA, this results in insertion of the fusion peptide into
the viral membrane (Korte et al. 1999; Weber et al. 1994).

The fourth test is to assess whether cells expressing the viral fusion
protein can fuse. Cell-cell fusion can be observed by light or fluores-
cence microscopy (Frey et al. 1995; Melikyan et al. 1997b; Mothes et al.
2000), or it can be scored with gene reporter assays that monitor inter-
actions of components from the fusing cells (Delos and White 2000;
Earp et al. 2003; Feng et al. 1996; Nussbaum et al. 1994). Although cell-
cell fusion assays are relatively simple to perform, the results do not
always correlate with virus-cell fusion or infection (Earp et al. 2003;
Lavillette et al. 1998; Schmid et al. 2000).

2.2

Receptor Activation at Neutral pH

Many enveloped viruses do not require low pH to fuse with target cells.
This has generally been established in controlled experiments using the
approaches described in Sect. 2.1. Viruses that can fuse at neutral pH in-
clude paramyxoviruses, herpesviruses, coronaviruses, poxviruses, and
most retroviruses (Table 1) (Hernandez et al. 1997; McClure et al. 1990;
Stein et al. 1987; Taguchi and Matsuyama 2002). The fusion proteins of
these viruses are activated via specific interactions with one or more re-
ceptors in the target cell membrane (Hernandez et al. 1996; Hunter 1997;
Stein et al. 1987). Viruses that can fuse at neutral pH are thought to do
so at the plasma membrane. However, they may also be able to fuse with
neutral-pH intracellular compartments (e.g., caveosomes) that can be
accessed through newly recognized endocytic pathways (Pelkmans and
Helenius 2003; Shin and Abraham 2001) (see also the chapter by
Sieczkarski and Whittaker, this volume). It is important to note, howev-
er, that viruses that can fuse at neutral pH may also possess the ability
to fuse at low pH (Earp et al. 2003; Fackler and Peterlin 2000). To date,
neutral-pH fusion has been found to display a sharp temperature thresh-
old, with little or no fusion occurring at T<20°C.

2.3

"Two-Step" Activation

Recently, a third model was proposed for the activation of alpharetro-
viruses. In this model, activation of the alpharetroviral Env begins with
receptor binding at neutral pH (at T>22°C) but is only complete after
exposure to low pH (Mothes et al. 2000). The role of low pH in this

L.J. Earp et al.

"two-step" model is derived from two key observations: (1) The continu-
ous presence of endosomal acidification inhibitors prevents production
of alpharetroviral reverse transcripts, and (2) cells expressing Env and
cells expressing the viral receptor only form large syncytia after expo-
sure to low pH (Mothes et al. 2000). Our recent work indicates that al-
pharetrovirus fusion can proceed to the lipid mixing stage at neutral
pH (Earp et al. 2003), and that receptor binding and low pH sequen-
tially induce distinct conformational changes in the alpharetrovial Env
(Matsuyama et al. 2004). Current work is now focused on determining
the precise role of low pH in the fusion cascade.

Classification of Fusion Proteins Based on Structural Criteria

All viral fusion proteins contain a relatively large ectodomain, generally
a single transmembrane domain, and all contain a cytoplasmic tail. So
far, two major groups (class I and class II) have been defined based on
structural criteria (Heinz and Allison 2001; Lescar et al. 2001) (Tables 1
and 2).

Class I fusion proteins are synthesized as precursors that are cleaved
into two subunits by host cell proteases. In some cases (e.g., influenza
HA), the two subunits remain associated through a disulfide bond; in
others (e.g., HIV Env), the two subunits remain associated through non-
covalent interactions. The proteolytic processing event that generates
the two subunits is critical, as it creates the metastable state of the fusion
protein (Chen et al. 1998). Class I fusion proteins exist as relatively long
trimeric spikes in both their metastable and activated states. In their
metastable states, they project perpendicularly to the viral membrane.
The activated forms of the fusion subunits of known class I fusion pro-
teins are highly a-helical (Skehel and Wiley 2000), and the final lowest-
energy (which we will refer to as "postfusion") forms (Fig. 1) contain
"six-helix bundles" (Bullough et al. 1994; Carr and Kim 1993). All six-he-
lix bundles contain a relatively long (65-115 A) central N-terminal tri-
meric coiled-coil. Some (e.g., HIV Env, SIV Env, and paramyxovirus F)
form six-helix bundles that extend to their membrane proximal ends
[i.e., three C-terminal helices (Fig. 1 A, green) pack in the grooves of the
central coiled-coil (Fig. 1A, blue)]. Others display a mixture of helical
and nonhelical segments that pack into the grooves of the central coiled-
coil. For example, the HA2 subunit of influenza HA contains a relatively
small six-helix bundle (Fig. 1, green/blue) at its membrane distal end,

The Many Mechanisms of Viral Membrane Fusion Proteins

Table 2. Class I vs. class II viral membrane fusion proteins

Property

Class I

Class II

Type of integral membrane protein

Type I

Type I a

Synthesized as

Inactive precursor

Inactive precursor b

Exist on virion in

Metastable state

Orientation in virion (to membrane)

Perpendicular

Parallel

Converted to metastable state by

Proteolytic processing

within fusion protein

of an associated

precursor

protein

No. of sub units in fusion protein

Major secondary structure of fusion

a-Helix c

/?-Sheet

subunit

Activated to fusogenic form by

Low pH or cell
receptor(s) d

LowpH

Oligomeric state of metastable protein

Trimer

Dimer

Oligomeric state of fusion active

Trimer

protein

Location of fusion peptide

N-terminal or internal

Internal loop

Structure of final fusogenic form

Trimer of hairpins

(coiled-coil)

(non-coiled-coil)

a The TBE E glycoprotein has two membrane anchoring segments near its C-termi-
nal end [Heinz and Allison (2001) Curr Opin Microbiol 4:450-5].

b Known class II fusion proteins are activated by proteolytic cleavage of an accesso-
ry protein.

c The postfusion forms of all known class I fusion proteins are a-helical. The fusion
subunit of metastable influenza HA is also highly a-helical, and this appears to be
the case for a paramyxovirus F protein [Chen et al. (2001a) Structure 9:255-66].
Comparable information is not available for the metastable forms of other class I
fusion proteins.

d In the case of paramyxoviruses, the receptor binding protein relays the informa-
tion of receptor binding to the fusion subunit [Lamb 1993; Colman and Lawrence
2003]

followed by an extended chain (Fig. 1, yellow) that packs in the groove
and extends to the N-terminal (membrane proximal) end of its central
coiled-coil (Fig. IB). Because of these variations, the postfusion forms
of class I fusion proteins are often referred to as "trimers of hairpins"
(Eckert and Kim 2001).

The general structure of class II fusion proteins is quite different from
that of class I fusion proteins. A well- characterized example is the enve-
lope glycoprotein (E) of tick-borne encephalitis (TBE) virus. During bio-
synthesis, TBE E and a second viral membrane glycoprotein, the precur-
sor to the membrane protein (prM), form heterodimers. As virions ma-

L.J. Earp et al

Fig. 1. Structures of the postfusion forms of SIV Env (A) and influenza HA (B). A
NMR structure of the postfusion form of SIV Env gp41 subunit (PDB accession
number 2EZO). B Crystal structure of the postfusion form of influenza HA2 subunit
(PDB accession number 1QU1). Coiled-coil regions are blue. C-terminal helices are
green. For influenza HA2, the C-terminal extended region is yellow. N and C indicate
the points where the fusion peptide and the transmembrane domain, respectively,
attach

ture, a host cell protease cleaves prM, resulting in reorganization of pro-
teins on the viral surface (Allison et al. 1995). After prM cleavage, the E
proteins exist as metastable homodimers. The ectodomains of the dimer
are oriented antiparallel to one another. In further contrast to the tri-
meric class I fusion protein spikes, the ectodomains of the E homodimer
lie parallel to the viral membrane and close to the surface. The TBE E
protein is composed mostly of /2-strand structure (Heinz and Allison
2001; Rey et al. 1995). The architecture of the Semliki Forest virus (SFV)
spike, another well-characterized class II fusion protein, is similar to
that of TBE E, but in this case, the metastable oligomer is a heterodimer
of two membrane-anchored proteins, El and E2, with an associated
small protein (E3).

Examples of Fusion Activation Mechanisms

In Sects. 4.1-4.4, we discuss a few examples of viral fusion proteins that
employ different fusion mechanisms in more detail. These will include

The Many Mechanisms of Viral Membrane Fusion Proteins

examples of class I and class II fusion proteins, activated by low pH or
by receptor interactions at neutral pH.

4.1

Influenza HA (Class I Fusion Protein, Low pH)

High-resolution structures are available for both the complete native
(metastable) (Wilson et al. 1981) and activated (Bullough et al. 1994; Chen
et al. 1999) forms of the influenza HA. On the viral surface, HA exists as a
trimer of heterodimers (Fig. 2A). Each heterodimer consists of HA1,
which contains the receptor binding domain (Fig. 2, gray), and HA2,
which contains the fusion peptide (Fig. 2, red) and the transmembrane
domain (located at the C-terminus). In the native (neutral pH) structure,
the fusion peptide is buried within the HA oligomer. Three long helices,
one from each monomer, come together to form the triple-stranded
coiled-coil of the metastable trimer (Fig. 2A and B, blue and green).

Fig. 2A-D. Low-pH-induced conformational changes within influenza HA. HA1 is
depicted in gray. The fusion peptide is red (HA2 residues 1-24). The coiled-coil is
blue, with the C-terminal helix colored green. The C-terminal extended region is yel-
low. The transmembrane domain (not shown) attaches to the C-terminal end, indi-
cated by "C", of HA2. A model for conformational changes: A In the native, metasta-
ble, structure of HA, the fusion peptides are buried within the trimer interface. HA1
acts as a clamp to hold HA2 in a metastable state. HA2 is largely shielded by HAL
To illuminate the HA2 core, we have cartooned the portion of HA1 that covers HA2
as a simple {gray) line. B On exposure to low pH, the HA1 headgroups separate, al-
lowing expulsion of the fusion peptide. C A loop-to-helix transition causes the fusion
peptide to be repositioned to one end of HA2, where it can bind to the target mem-
brane. D A helix-to-loop transition causes the C-terminal helix and the C-terminal
extended region to reverse direction and bind to the grooves of the coiled-coil in an
antiparallel orientation

L.J. Earp et al.

On exposure to low pH, HA undergoes dramatic conformational
changes. The globular head domains separate, releasing the clamp that
holds HA2 in its metastable state (Fig. 2B). As a result, the fusion pep-
tide is exposed (Fig. 2C, red) at the top of an extended triple-stranded
coiled-coil, in a position where it can interact with the target membrane.
A helix-to-loop transition causes a short helix (Fig. 2D, green) and the
C-terminal extended region (yellow) to flip up and run antiparallel to
the central coiled-coil (Bullough et al. 1994). As a result, the fusion pep-
tide and transmembrane domain are brought into close proximity at the
same end of the molecule (Fig. 2D).

Many regions of HA are important for fusion. The fusion peptide is
critical for hydrophobic attachment of the virus to the target membrane
(Sect. 6.1). Mutations that prevent (1) globular head domain separation
(Godley et al. 1992; Kemble et al. 1992), (2) the "B-loop"-to helix transi-
tion (Gruenke et al. 2002; Qiao et al. 1998), or (3) the C-terminal ex-
tended region from packing into the grooves of the final coiled-coil
(Borrego-Diaz et al. 2003; Park et al. 2003) ablate the ability of HA to
reach the lipid mixing stage of fusion. In our model (Gruenke et al.
2002), conversion of HA to a prehairpin intermediate (Fig. 2C) allows
HA to bind to the target membrane. Further conversion to the hairpin
structure (Fig. 2D) then drives the formation and opening of a fusion
pore.

4.2

HIV Env (Class I Fusion Protein, Neutral pH)

Like influenza HA, HIV Env is synthesized as a single-chain precursor
and cleaved during biosynthesis to yield gpl20 and gp41. Native (meta-
stable) HIV Env is a trimer of the heterodimers of gpl20 (the receptor
binding subunit) and gp41 (the fusion subunit). Env is activated for
fusion (at neutral pH) after sequential binding to CD4 and a coreceptor
(a chemokine receptor). Binding of Env to CD4 causes conformational
changes in Env that permit binding to the coreceptor. After coreceptor
binding, additional conformational changes occur in Env that lead to fu-
sion (Eckert and Kim 2001).

Crystal structures exist for the core of the gpl20 subunit (Kwong et
al. 1998) as well as for the postfusion (Fig. 3, Step 6) form of gp41 (Chan
et al. 1997; Weissenhorn et al. 1997). However, there is not yet a crystal
structure of the native (metastable) Env trimer. Therefore, a detailed pic-
ture of HIV Env activation via receptor interaction is not available. We
presume that the first steps of Env activation are separation of the

The Many Mechanisms of Viral Membrane Fusion Proteins

~wr

Fig. 3. Model of HIV fusion. Env exists as a trimer in the surface of the native viral
membrane, with fusion peptides (red) presumably buried within the trimer inter-
face. SU domains (pictured as gray globular domains at the top of the trimer) pro-
vide the receptor-binding function. For clarity, SU domains are omitted after Step 1.
Target cell receptors are not pictured in this model. On exposure to receptor and
coreceptor at T >22°C and neutral pH, Env undergoes conformational changes that
result in exposure of the fusion peptides (Step I), which then insert into the target
membrane (Step 2). Multiple Envs may cluster (Step 3) to form a fusion site. Addi-
tional conformational changes (Steps 4 and 5) lead to the formation of a six-helix
bundle, resulting in hemifusion (Step 5) (defined as mixing of the outer leaflets of
the viral and cellular membranes). Eventually a fusion pore forms (Step 6) and en-
larges (not shown)

globular head domains, expulsion of the fusion peptide, and extension
of gp41 into a prehairpin intermediate (Fig. 3, Step 1). Several lines of
evidence indicate the existence of the prehairpin intermediate. For ex-
ample, peptide analogs of the C-terminal helix (Fig. 3, green) strongly
inhibit HIV fusion and infection (Chan and Kim 1998; Kilby et al. 1998).
Also, a synthetic peptide corresponding to the C-terminal helix coim-
munoprecipitates with HIV Env after engagement of receptors (Furuta
et al. 1998; He et al. 2003). The C-terminal helix then packs, in an anti-
parallel fashion, into the groove of the N-terminal coiled-coil (Fig. 3,
Step 5). Because the C-terminal helices of gp41 extend along the entire
length of the N-terminal coiled-coil, this packing would bring the fusion
peptide and transmembrane domain very close together. The transition
to the six-helix bundle drives membrane merger (Melikyan et al. 2000a).
Moreover, complete six-helix bundles are needed to form "robust" fu-
sion pores (Markosyan et al. 2003).

As mentioned above, HIV studies, primarily using epitope accessibili-
ty assays, have indicated that engagement of HIV receptors induces con-

L.J. Earp et al.

formational changes in gpl20 and gp41 (Eckert and Kim 2001; Xiang et
al. 2002). A remaining issue for all receptor-activated viral fusion pro-
teins is how information is transmitted (after receptor binding) through
the receptor binding subunit to the fusion subunit. Such transmission is
essential to allow rearrangements in the fusion subunit (e.g., six-helix
bundle formation) that drive fusion. For HIV, part of the mecha-
nism may involve reduction of one or more disulfide bonds in gpl20
(Abrahamyan et al. 2003; Barbouche et al. 2003; Fenouillet et al. 2001;
Gallina et al. 2002). In murine retroviral Envs, a proline-rich hinge re-
gion appears to relay receptor binding information from the N-terminal
to the C-terminal region of the receptor binding subunits (SU) (Barnett
and Cunningham 2001; Lavillette et al. 2001). Because the proline-rich
region of SU is linked to TM by a disulfide bond (Pinter et al. 1997), this
may provide a relay system to trigger conformational changes in the fu-
sion subunit. Clearly, the molecular pathways by which receptor-activat-
ed fusion proteins change from their metastable to their activated forms
need to be defined.

In Fig. 3, we show a working model for HIV Env-mediated fusion. It
is derived in part from studies with influenza HA, and it is similar to
other HIV fusion models (Eckert and Kim 2001). Our hypothesis is that
all class I fusion proteins will employ similar mechanisms. We note,
however, that even in the case of influenza HA, alternate models are still
entertained (see Fig. 2 in Jahn et al. 2003). Furthermore, others have
suggested that different class I fusion proteins may use fundamentally
different mechanisms (Chen et al. 2001a).

The features that we predict will be common to the fusion mecha-
nisms of all class I fusion proteins (Fig. 3) include: (1) conversion from
a metastable state to an activated state, (2) exposure and repositioning
of the fusion peptide for binding to the target bilayer, (3) recruitment of
several activated fusion proteins to a fusion site (Blumenthal et al. 1996;
Danieli et al. 1996; Markovic et al. 2001; Markovic et al. 1998), and (4)
subsequent conformational changes that result in close apposition of the
fusion peptide and the transmembrane domain.

4.3

Paramyxovirus F Proteins (Class I Fusion Protein, Neutral pH,
Attachment Protein Assisted)

The viral fusion proteins that have thus far been discussed in detail con-
tain a receptor binding domain (e.g., the gpl20 subunit of HIV Env)
within the fusion protein spike. In other cases, the receptor binding do-

The Many Mechanisms of Viral Membrane Fusion Proteins 37

main resides in a separate viral spike. Paramyxoviruses have an attach-
ment protein spike and a separate fusion (F) protein spike. Most, but
not all, paramyxoviruses require both the attachment protein and the F
protein for fusion (Bagai and Lamb 1995; Paterson et al. 2000). In most
cases, the attachment protein must come from the same paramyxovirus
as the fusion protein (Bossart et al. 2002). In the few cases in which the
F protein is sufficient, fusion is enhanced if the attachment protein is
also expressed (Bagai and Lamb 1995). The need for the attachment pro-
tein can be overcome by mutations in the F protein (Paterson et al. 2000;
Seth et al. 2003) or by conducting fusion reactions at T>37°C (Paterson
et al. 2000; Wharton et al. 2000). Paramyxovirus fusion proteins thus re-
present special cases of receptor-activated fusion proteins, in which re-
ceptor activation is communicated from one viral spike glycoprotein to
another.

F proteins are proteolytically cleaved during biosynthesis to generate
two disulfide-bonded subunits, Fi and F 2 (Begona Ruiz-Arguello et al.
2002; Gonzalez- Reyes et al. 2001; Lamb 1993), found as metastable tri-
mers of dimers (Baker et al. 1999) on virions. It has been suggested that
binding of the attachment protein to a host cell receptor causes confor-
mational changes in this protein, which in turn cause activating confor-
mational changes in the metastable F protein (Lamb 1993; Russell et al.
2001; Takimoto et al. 2002). The exact mechanism by which attachment
proteins activate F proteins is not known, but several groups have pro-
vided evidence for cross talk between attachment and F proteins (Bos-
sart et al. 2002; Deng et al. 1999; McGinnes et al. 2002; Stone-Hulslander
and Morrison 1997; Takimoto et al. 2002; Yao et al. 1997).

The post-fusion form of the F protein from the paramyxovirus SV5
contains a six-helix bundle (Baker et al. 1999). Similar to HIV Env (He
et al. 2003; Kilby et al. 1998; Munoz-Barroso et al. 1998) and other retro-
viral fusion proteins (Earp et al. 2003; Netter 2002), peptide analogs of
the N- and C-terminal helices of paramyxovirus six-helix bundles are
potent inhibitors of fusion and infection (Bossart et al. 2002; Joshi et al.
1998; Lambert et al. 1996; Young et al. 1999). As is also the case for HIV
Env (Markosyan et al. 2003; Melikyan et al. 2000a), a recent study
showed that conversion of the SV5 F protein to a six-helix bundle drives
membrane fusion (Russell et al. 2001).

Issues yet to be addressed for paramyxoviruses are the structure of
the complete native (metastable) F trimer and how it is converted to its
activated form. The first glimpses at the metastable and postfusion states
of the F trimer came from EM observations of the respiratory syncytial
virus (RSV) F protein. Preparations of purified recombinant F protein

L.J. Earp et al.

contained both cone-shaped rods and "lollipop"-shaped structures. On
storage, there appeared to be a shift from the cone-shaped to the "lol-
lipops-shaped structures (Calder et al. 2000). Examination of F com-

plexed with specific monoclonal antibodies suggested that the "lollipop"
structures contained six-helix bundles composed of N- and C-terminal
heptad repeats (Calder et al. 2000).

A high-resolution structure of an F protein ectodomain was recently
presented (Chen et al. 2001a). The protein used for the analysis contained
a mixture of precursor F and proteolytically cleaved F. It also apparently
lacked the second heptad repeat, which forms the C-helix in the postfu-
sion form. This trimeric F protein structure is fundamentally different
from that of influenza HA; the N-terminal end of its coiled-coil is posi-
tioned near the viral membrane end of the molecule (i.e., opposite the
orientation of the coiled-coil in the metastable HA trimer). If this F pro-
tein structure represents the native metastable F trimer, then it suggests a
mechanism of fusion activation for F fundamentally different from that
for HA (Chen et al. 2001a). Additional work is needed to test this idea.

4.4

TBE E and SFV El (Class II Fusion Proteins, Low pH)

All known class II fusion proteins are activated by low pH. However, the
mechanism by which class II fusion proteins are initially activated is
quite different than the mechanism by which class I fusion proteins are
initially activated. For example, the ectodomain of the TBE glycoprotein
forms an antiparallel dimer that lies parallel and close to the viral mem-
brane (Fig. 4B). At low pH, the TBE E homodimer converts to an E ho-
mo trimer (Allison et al. 1995; Heinz and Allison 2001; Stiasny et al.
2001). This transformation is thought to occur in two steps: dissociation
of the E homodimer, followed by reassociation of E trimers (Stiasny et
al. 1996). Membrane binding occurs after dimer dissociation and pro-
motes the formation of E homotrimers (Stiasny et al. 2002). Homotrimer
formation may involve interactions between a-helices in the stem region
of the E protein (Allison et al. 1999).

The SFV fusion protein also converts from a dimer to a trimer during
fusion activation. On native virions, El exists as a tight heterodimeric
complex with a second membrane protein, E2. On exposure to low pH,
El dissociates from E2, changes conformation, and forms highly stable
El homotrimers (Ahn et al. 1999; Kielian 1995; Wahlberg et al. 1992;
Wahlberg and Garoff 1992). During this process, El binds hydrophobi-
cally through its fusion peptide to target membranes and mediates fu-

The Many Mechanisms of Viral Membrane Fusion Proteins

m!$Mm**

^«T>V! iminTivniTHTnivi

'nii'TT'n

>n*m ■n-T*T-> , n

*% 1 % **%* »~% « %% M 4

Fig. 4. Structure and cartoon of conformational changes of the TBE E protein. A
Crystal structure of TBE E (PDB accession number 1SVB). The fusion peptide is red.
Domains I, II, and III are pink, blue, and green, respectively. Disulfide bonds are
black. B Cartoon depicting possible rearrangements during the dimer to trimer tran-
sition upon exposure to low pH (Allison et al. 1995). Each of the three dimers {blue,
green, yellow, left) supplies one monomer (light shaded subunits) to the homotrimer
(right). The organization of the dimers is as found in TBE recombinant subviral par-
ticles (Ferlenghi et al. 2001); it may represent an intermediate arrangement (from
that on native virions) found during fusion activation (Kuhm et al. 2002). Note that
other possibilities for the dimer to trimer transition exist (for example involving rel-
ative movements of domains about hinge regions). For very recent developments re-
garding the fusion mechanism of class II fusion proteins, see Bressanelli et al.
(2004), Gibbons et al. (2004) and Modis et al. (2004)

sion. Similar to TBE E, it appears that binding to the target bilayer fos-
ters formation of the activated El homotrimer (Kielian 1995; Kielian et
al. 2000); the fusion peptide and the transmembrane domain of El ap-
pear to be important for El homotrimer formation (Kielian et al. 1996,
2000; Sjoberg and Garoff 2003). Thus both class I and class II viral fu-
sion proteins appear to function as trimers during fusion. It has been
proposed that activated TBE E (Helenius 1995) and SFV El stand up as
trimeric spikes and present their fusion peptides to the target membrane
(Fig. 4B, right). This would be analogous to Fig. 3, Step 1. If this occurs,

L.J. Earp et al.

then the spike would have to refold to bring the viral and cellular mem-
branes together (e.g., analogous to Steps 4 and 5 in Fig. 3). Very recent
evidence indicates that this is, indeed, the case (Bressanelli et al. 2004;

Gibbons et al. 2004; Modis et al. 2004).

Membrane Dynamics During Fusion

Thus far we have focused on the conditions that elicit viral fusion reac-
tions and the conformational changes in viral fusion proteins necessary
for fusion. However, it is the viral and cellular bilayer membranes that
merge during fusion. Lipid bilayers are stable structures that do not fuse
spontaneously. Fusion proteins have evolved to catalyze the necessary
lipid rearrangements. We now review a lipid rearrangement model and
focus on the roles of different regions of viral fusion proteins in chore-
ographing the structural changes that the membranes undergo through-
out the fusion cascade (Fig. 3).

The favored model for the lipid transition state during membrane fu-
sion is the stalk model. In this model, two opposing membranes bend
toward each other, creating "dimples" (when viewed from the trans sur-
face) or "nipples" (when viewed from the cis surface) (Fig. 3, Step 4).
Nipples continue to bend until they meet. The two cis leaflets then
merge, creating a lipid stalk (see Fig. 2 in Kozlovsky and Kozlov 2002)
that proceeds to a state of local hemifusion (Fig. 3, Step 5). In a second
step, transient fusion pores form, which give rise to stable pores (Fig. 3,
Step 6).

The first direct visualization of a lipid stalk intermediate was achieved
by electron diffraction studies of the effect of sequential dehydration on
lipid bilayers composed of a lipid that has negative spontaneous curva-
ture (Yang and Huang 2002). The stalk intermediate was stable at inter-
mediate relative humidities. The results suggested that both the forma-
tion of a lipid stalk and its transition to a conformation that can be
equated with pore formation require external forces.

Cellular membranes do not have spontaneous negative curvature and
are highly hydrated. Membrane curvature can be promoted by introduc-
ing defects into the contacting bilayers. Thus roles for the fusion protein
include pulling the fusing bilayers toward one another (dimpling), dehy-
drating the membranes, and creating membrane defects that lower the
energy barrier for stalk and pore formation. Two intermediates in HIV
fusion have been trapped: one in which the two membranes are joined

The Many Mechanisms of Viral Membrane Fusion Proteins 41

by activated Envs, but are not yet fused (Melikyan et al. 2000a), and one
in which small, "labile" pores have formed that can either expand into
stable, "robust" pores or return to the prefusion state (Markosyan et al.
2003). These observations suggest a role for the fusion protein in forma-
tion and stabilization of both the fusion stalk and the fusion pore.

The mechanism by which a small pore enlarges is not known. How-
ever, several possibilities have been proposed. One is that the initial fu-
sion pore is formed by a small number of activated fusion proteins. Ad-
ditional activated fusion proteins then move into the fusion site to but-
tress and stabilize the pore, thereby allowing it to expand (Kozlov and
Chernomordik 2002). Another possibility is that multiple small fusion
pores coalesce to form larger ones. This was supported by EM visualiza-
tion of HA-mediated fusion, in which multiple dimples/nipples were ar-
ranged circularly and lipid fragments were seen at the center of a fusion
ring (Kanaseki et al. 1997).

Membrane-Interacting Regions of Viral Fusion Proteins

As discussed above, roles for the fusion protein in the fusion cascade
(Fig. 3) include pulling the fusing bilayers toward one another (dim-
pling) and creating membrane defects that lower the energy barriers for
stalk formation and fusion pore opening/enlargement. The fusion pep-
tide and the transmembrane domain must remain stably associated with
the target and viral membranes, respectively, for fusion to occur. Once
the fusion peptide is stably associated with the target bilayer (Fig. 3, Step
2), we envision that rearrangements in the fusion protein ectodomain
that bring the fusion peptide and transmembrane domains close togeth-
er (Fig. 3, Step 4) result in dimpling of membranes toward one another.
In addition to serving as critical membrane anchors, the fusion peptide
and the transmembrane domain likely create membrane defects that fa-
cilitate the next stages of fusion. Here, we review information about the
structure and function of the fusion peptide and the transmembrane do-
main during fusion. We also review evidence that juxtamembrane se-
quences, on both sides of the transmembrane domain, participate in fu-
sion.

L.J. Earp et al.

6.1

The Fusion Peptide

Fusion peptides are relatively apolar sequences that interact with mem-
branes and are central to viral fusion reactions (Martin and Ruysschaert
2000; Martin et al. 1999; Skehel et al. 2001; White 1990). They have been

N-terminal
Class I

Influenza HA2 :
Sendai PI:

, Syn, Pis

HIV gp41:

GLFGAIAGFIENGWEG
FFGAVIGTIALGVATA
FLGFLLGVGSAIASGV
AAIGALFLFGLGAAGSTMGAA

Internal
Class I

Ebola GP:

ASLV gp37

GAAIGLAWIPYFGPAA
IFAS ILAPGVAAAQAL

Class II

SFV El:

TBE E:

DYQCKVYTGVYPFMWGGAYCFCD
DRGWGNHCGLFGKGSIVA

unclassified VSV G:

QGTWLNPGFPPQSCGYATV

Fig. 5A, B. Characteristics of viral fusion peptides. A Selected viral fusion peptide se-
quences. N-terminal (Skehel et al. 2001) and internal (Delos et al. 2000) fusion pep-
tide sequences are aligned according to their first noncharged residue. B Model of
HA fusion peptide structure in target membrane at pH 5 (adapted from Tamm et al.
2002). The fusion peptide (red) resides in the target membrane in a kinked structure
composed of two a-helices, each penetrating the outer leaflet. The glycine ridge is
depicted by a yellow box, the hydrophobic interior face by cyan ovals, and the sur-
face charged residues by blue squares. "C" denotes the direction of the HA2 ectodo-

main

The Many Mechanisms of Viral Membrane Fusion Proteins 43

classified as N-terminal or internal depending on their location within
the fusion subunit (Table 1). Although fusion peptides are highly con-
served within each virus family, there is little sequence similarity be-
tween fusion peptides of different families (Fig. 5A). Generally, however,
fusion peptides contain a high percentage of glycines and/or alanines,
as well as several critical bulky hydrophobic residues (Martin and
Ruysschaert 2000; Martin et al. 1999; Skehel et al. 2001; Tamm and Han
2000; Tamm et al. 2002).

6.1.1

Structure of N-terminal Fusion Peptides

A significant body of work has emerged on the structure and function of
synthetic fusion peptides (Martin and Ruysschaert 2000; Martin et al.
1999; Skehel et al. 2001; Tamm and Han 2000; Tamm et al. 2002). Syn-
thetic fusion peptides are disordered in solution but ordered (a-helix
and/or y8-sheet) when they associate with membranes. The N-terminal
fusion peptides that have been studied insert into membranes at oblique
angles and do not penetrate the inner leaflet of the membrane. In gener-
al, mutations that abrogate fusion reduce the ability of synthetic fusion
peptides to insert at oblique angles and to disrupt membranes (Martin
et al. 1999). Contradictory conclusions on the precise structure of syn-
thetic fusion peptides in membranes likely stem from the general low
solubility of the peptides in aqueous solution and the different experi-
mental methods employed (Tamm et al. 2002).

To circumvent solubility problems, a polar sequence was added to the
C-terminal end of the influenza HA fusion peptide, rendering it soluble
in both aqueous and hydrophobic environments (Han et al. 2001). At pH
5, the HA fusion peptide consists of an N-terminal helix, a kink, and a
short C-terminal helix (Fig. 5B). Both the N- and C-terminal helices pen-
etrate the outer leaflet of the target bilayer. The kink remains at the
phospholipid surface; the interior (lipid-facing surface) of the kink is
lined with hydrophobic residues. The conserved glycines form a ridge
along the outer face of the N-terminal helix. Three charged residues are
also found on the outer face (Fig. 5B). An HA in which the conserved
glycine at the beginning of the fusion peptide (Glyl) has been changed
to valine cannot mediate fusion. If Glyl is changed to serine, HA medi-
ates only hemifusion or only forms small nonexpanding fusion pores
(Qiao et al. 1999; Skehel et al. 2001). Interestingly, these mutant fusion
peptides have membrane-associated structures and orientations signifi-
cantly different from those of the wild-type fusion peptide (Li et al.

L.J. Earp et al.

2003). Simulations suggested similar membrane penetrating orientations
for the HIV fusion peptide and two fusion-defective mutants (Kamath

and Wong 2002).

6.1.2

Structure of Internal Fusion Peptides

In addition to a significant number of apolar residues, many internal
fusion peptides contain a conserved proline at or near their centers
(Fig. 5A). Mutagenesis of this proline in the avian sarcoma/leukosis vi-
rus (ASLV) EnvA fusion peptide suggested that it stabilizes a /?-turn
(Delos et al. 2000). This, coupled with the observation that mutating two
cysteines that flank the fusion peptide abolishes fusion activity (Delos
and White 2000), suggested that the internal EnvA fusion peptide exists
as a looped structure stabilized by a disulfide bond. The ability of the
Ebola virus fusion protein, which also contains an internal fusion pep-
tide, to support infection was similarly inhibited when its central proline
and flanking cysteines were mutated (Ito et al. 1999; Jeffers et al. 2002).
A similar mutation of a proline within the predicted turn segment of the
candidate fusion peptide of VSV G also significantly decreased fusion
and abolished infectivity (Fredericksen and Whitt 1995). The idea of
loop structures for internal fusion peptides is further supported by the
known looped structure of the TBE E and SFV EI fusion peptides (Rey et
al. 1995; Allison et al. 2001; Lescar et al. 2001). In some cases, two or
more noncontiguous sequence loops may function as a collective fusion
peptide (Gaudin et al. 1999a; Li et al. 1993).

Like N-terminal fusion peptides, internal fusion peptides contain a
significant number of glycines and hydrophobic residues (Fig. 5A).
Changing either of two glycines within the SFV El fusion peptide to ala-
nines altered the pH threshold for fusion, and changing one of the gly-
cines to aspartic acid abolished fusion (Duffus et al. 1995; Kielian et al.
1996). Alteration of hydrophobic residues at the beginning, middle, or
end of the (internal) ASLV EnvA fusion peptide to charged residues im-
paired the ability of EnvA to mediate fusion (Hernandez and White
1998). Similarly, a tryptophan and a glycine are critical for Ebola GP-me-
diated infection (Ito et al. 1999). Also, a bulky hydrophobic residue is
needed at the tip of the TBE E fusion peptide loop (Rey et al. 1995)
(Fig. 4A, red). Collectively, these results suggest that internal fusion
peptides function as loops that require a mixture of hydrophobic and
flexible residues, similar to those found in N-terminal fusion peptides.

The Many Mechanisms of Viral Membrane Fusion Proteins 45

6.1.3

Roles of Fusion Peptides

Fusion peptides appear to act at several steps along the fusion pathway.
As demonstrated by mutants in which apolar fusion peptide residues
were changed to charged residues (Freed et al. 1992; Gething et al. 1986;
Hernandez and White 1998; Schoch and Blumenthal 1993), fusion pep-
tides clearly play an important role in anchoring the fusion protein to
the target membrane (Fig. 3, Step 2). The energy provided by inserting
the fusion peptides of a single HA trimer into a membrane would be suf-
ficient to initiate stalk formation (Gunther-Ausborn et al. 2000). The
fusion peptide may also assist in creating the stalk by displacing water
from the lipid-water interface, thus decreasing the repulsive force be-
tween the two fusing membranes (Tamm and Han 2000). Fusion pep-
tides may also function in fusion pore opening. In support of this possi-
bility is the observation that an HA mutant in which Glyl was changed
to serine mediates extensive lipid, but not content mixing (Qiao et al.
1999). Furthermore, defects in syncytium formation and infectivity were
observed for HIV Env harboring the mutation V2E in its fusion peptide
(Freed et al. 1992). Biophysical studies comparing a synthetic fusion
peptide harboring this mutation with the wild-type peptide suggested a
requirement for fusion peptide aggregation in the creation of the HIV
fusion pore (Kliger et al. 1997; Pereira et al. 1995).

6.2

The Transmembrane Domain

Studies with chimeric fusion proteins have suggested that the transmem-
brane domains of some viral fusion proteins do not require a specific se-
quence to support fusion (Armstrong et al. 2000 and references therein).
In contrast, studies with glycosylphosphatidylinositol (GPI)-anchored
fusion proteins have demonstrated that there is a strict requirement for
a proteinaceous membrane anchor for fusion proteins to efficiently me-
diate the transition from hemifusion to full fusion (Kemble et al. 1994;
Melikyan et al. 1997a; Tong and Compans 2000). There also appears to
be a minimum length for the fusion protein transmembrane domain to
be able to support this transition (Armstrong et al. 2000; West et al.
2001). Therefore, it has been suggested that fusion protein transmem-
brane domains must span both leaflets of the viral bilayer to mediate fu-
sion pore opening (Armstrong et al. 2000).

L.J. Earp et al.

The transmembrane domains of some fusion proteins appear to have
specific amino acid requirements for fusion function. For example, a
conserved positively charged residue in the middle of the transmem-
brane domains of certain retroviral Envs appears to be important for the
ability to mediate fusion and infection (Einfeld and Hunter 1994; Owens
et al. 1994; Pietschmann et al. 2000; West et al. 2001). Two glycine resi-
dues in the transmembrane domain of VSV-G appear to be important
for the transition from hemifusion to full fusion (Cleverley and Lenard
1998). Studies using a synthetic peptide corresponding to the mutant
VSV-G transmembrane domain (Dennison et al. 2002) suggested that
the VSV-G transmembrane domain lowers the energy barrier for fusion
and stabilizes the transient fusion pore, thereby promoting its conver-
sion to a stable fusion pore. Two glycines may allow the VSV-G trans-
membrane domain to adopt alternative conformations under different
conditions, and such flexibility may be important for function. The
transmembrane domain of HA from the Japan (Melikyan et al. 2000b),
but not the X:31 (Armstrong et al. 2000), strain of influenza appears to
require a glycine near the middle. An ability to adopt alternative confor-
mations was also invoked to explain the requirement for a proline near
the middle of the transmembrane domain of the murine leukemia virus
(MLV) Env glycoprotein (Taylor and Sanders 1999).

The observations that mutations in fusion peptides or transmem-
brane domains (Armstrong et al. 2000; Baker et al. 1999; Tamm et al.
2002) can impair the ability to mediate full fusion (Fig. 3, Step 6) have
suggested that both of these apolar domains function in the transition
from hemifusion to full fusion. Initially, the fusion peptide appears to in-
sert only into the outer leaflet of the target membrane (Tamm and Han
2000). It has been proposed that the transmembrane domain and the
fusion peptide, which are close to each other after membrane merger,
may interact to stabilize the fusion pore (Tamm et al. 2002; Zhou et al.
1997). If this is the case, the fusion peptide might span both leaflets of
the fused membrane in its final conformation (Tamm et al. 2002).

6.3

The Juxtamembrane Region of the Ectodomain

Several lines of evidence suggest that ectodomain sequences that lie just
before the transmembrane domains of certain viral fusion proteins may
be important for fusion. These sequences tend to have a high proportion
of tryptophans or other aromatic residues and are predicted to partition
into the interfacial regions of membranes (Suarez et al. 2000). Indeed,

The Many Mechanisms of Viral Membrane Fusion Proteins 47

synthetic peptides containing juxtamembrane ectodomain sequences
from HIV Env and Ebola GP partition into the interfacial region of tar-
get membranes (Saez-Cirion et al. 2003; Saez-Cirion et al. 2002; Schibli
et al. 2001; Suarez et al. 2000). Mutation of three tryptophans within this
region of HIV gp41 abrogated infection (Salzwedel et al. 1999), appar-
ently by inhibiting fusion pore enlargement (Munoz-Barroso et al.
1999). Extending the HIV gp41 C-terminal heptad repeat peptide to in-
clude the tryptophan-rich juxtamembrane ectodomain sequence ap-
peared to increase the potency of the peptide as an inhibitor of fusion
(Kliger et al. 2001). It was suggested that the extended heptad repeat
peptide was more potent because it could bind to two sites on HIV Env
(the N-terminal coiled-coil and a second, as yet unidentified site) (Kliger
et al. 2001). A likely effect of these peptides on late stages of fusion is to
prevent formation of a required structure in Env that provides addition-
al membrane destabilization. In this manner, partitioning of juxtamem-
brane sequences into the interfacial region of membranes may promote
the transition from a stalk intermediate to a fusion pore (see Sect. 5).

6.4

The Cytoplasmic Tail

A specific cytoplasmic tail sequence does not appear to be essential, but
it can modulate late stages of fusion. The cytoplasmic tail has been
shown to influence the transition from hemifusion to full fusion (Sakai
et al. 2002) or fusion pore enlargement (Dutch and Lamb 2001; Kozerski
et al. 2000) in some viruses. The mechanism by which cytoplasmic tails
may influence these later stages of fusion is not known. Some studies us-
ing synthetic peptides have suggested a direct interaction between the
cytoplasmic tail and the viral membrane (Chen et al. 2001b; Fujii et
al. 1992; Gawrisch et al. 1993; Haffar et al. 1991; Kliger and Shai 1997).
Others have shown that the cytoplasmic tail can influence the structure
of the ectodomain of the fusion protein (Aguilar et al. 2003; Edwards et
al. 2002).

The ability of the cytoplasmic tail to affect ectodomain structure is
most clearly manifested for those viral fusion proteins that harbor
fusion-suppressing sequences. These sequences have been found in the
fusion proteins of MLV (Ragheb and Anderson 1994), other type C
retroviruses (Bobkova et al. 2002), some lentiviruses (Kim et al. 2003;
Luciw et al. 1998), and a paramyxovirus F protein (Tong et al. 2002). The
cytoplasmic tail of MLV Env is cleaved during virus budding (Schultz
and Rein 1985). MLV Envs harboring uncleaved cytoplasmic tails do not

L.J. Earp et al.

induce fusion (Yang and Compans 1996). Although viruses lacking
fusion-suppressing sequences display increased cell-cell fusion, they are
more susceptible to neutralizing antibodies (Januszeski et al. 1997; Li et
al. 2001; Rein et al. 1994; Yang and Compans 1996) and are impaired in
their ability to sustain multiple rounds of infection (Cathomen et al.
1998; Freed and Martin 1996; Piller et al. 2000).

Acylation of cytoplasmic tails can also affect fusion, apparently at a
late stage. For example, a mutant HA from the Japan strain of influenza
in which three (normally palmitoylated) cysteine residues were mutated
appeared to fuse normally when monitored by dye redistribution assays
(Melikyan et al. 1997b). However, electrophysiological measurements re-
vealed that fusion pores formed by the mutant HA did not flicker like
those formed by wt-HA (Melikyan et al. 1997b). Similar mutations in
HA from the A/USSR/77 (H1N1) and A/FPV/Rostock/34 (H7N1) influ-
enza subtypes were shown, respectively, to inhibit syncytia formation
(Fischer et al. 1998) and the transition from hemifusion to full fusion
(Sakai et al. 2002). Palmitoylation of HIV Env was also shown to be im-
portant for Env incorporation into virions and for infectivity (Rousso et
al. 2000). Thus acylation of cytoplasmic tails appears to have multiple
effects on viral fusion reactions, the details of which are not completely
understood.

Rafts in Viral Membrane Fusion

Lipid rafts are plasma membrane microdomains that are enriched in
cholesterol and glycosphingolipids with saturated acyl chains. They are
organizational platforms for a variety of cellular functions including
sorting of membrane proteins and signaling (Brown and London 2000).
Although there is growing evidence that certain viruses employ rafts, or
raftlike membrane microdomains, during virus assembly (Suomalainen
2002), the question of whether these structures are required at the site of
fusion in the target cell is less clear. Here, we consider the role of rafts in
the fusion of two enveloped viruses, SFV and HIV. It is important to con-
sider whether cholesterol and/or sphingolipids are required for fusion
because they are found in lipid rafts, or if they serve some other pur-
pose. For example, cholesterol may interact directly with the fusion pro-
tein, thereby facilitating its insertion into the target membrane. Alterna-
tively, a need for cholesterol and sphingolipids could reflect an ability of
raft structures to concentrate viral receptors. A third possibility is that

The Many Mechanisms of Viral Membrane Fusion Proteins 49

the cholesterol imparts the membrane fluidity (or other biophysical
properties) needed to lower the energy barrier for fusion.

SFV requires cholesterol and sphingolipids in the target membrane
for fusion. These moieties enable the SFV spike protein to undergo con-
formational changes and bind to the target membrane (Ahn et al. 2002;
Kielian et al. 2000). In a recent study, it was shown that after hydropho-
bic association with target bilayers, the SFV glycoprotein ectodomain as-
sociates with membrane structures with properties similar to rafts.
However, careful studies using liposomes prepared with specific choles-
terol and sphingolipid analogs demonstrated that the cholesterol and
sphingolipid requirements in the target membrane did not correlate with
their ability to form lipid rafts (Ahn et al. 2002). A related conclusion
was drawn based on the fusion activities of both SFV and Sindbis virus
with liposomes (Waarts et al. 2002). For both viruses, the requirement
for cholesterol and sphingolipids in the target membrane appears to be
for insertion of the fusion peptide (Vashishtha et al. 1998).

In the case of HIV, several studies have suggested a need for raftlike
membrane microdomains for virus entry (Kozak et al. 2002; Popik et al.
2002). Depleting plasma membrane cholesterol from target cells resulted
in reduced levels of virus infectivity or cell-cell fusion. Other studies
have concluded that rafts are not necessary for HIV entry (Percherancier
et al. 2003; Viard et al. 2002). In one study, depleting cholesterol from
cells that express low levels of virus receptors inhibited HIV Env-medi-
ated cell-cell fusion, but depleting cholesterol from cells that express
high levels of virus receptors did not (Viard et al. 2002). Therefore, it
was concluded that rafts per se are not needed for fusion. Rather, the
presence of raftlike structures in the plasma membrane may concentrate
virus receptors. Previous work has shown that a critical density of HIV
receptors is required for fusion and infection (Reeves et al. 2002). Clear-
ly more work is needed to clarify the role of rafts in virus -cell fusion
and entry.

Inhibitors of Viral Fusion

It has recently become apparent, largely because of the success of T-20
in the inhibition of HIV infection in patients (Jiang et al. 2002; Kilby et
al. 1998), that fusion is a good target for antiviral intervention. This was
originally conceptualized because fusion is an essential early step in the
virus infectious cycle, it happens in an exoplasmic space, and strategies

L.J. Earp et al.

can be designed to inhibit fusion without interfering with host cell pro-
teins. Some fusion inhibitors function by inhibiting six-helix bundle for-
mation. Others function by preventing earlier conformational changes
in viral fusion proteins.

8.1

Inhibition of Helix Bundle Formation

The peptide T-20 (also known as Fuzeon) corresponds to the C-termi-
nal helix of HIV Env (Fig. 3, green). T-20 works by preventing six-helix
bundle formation. T-20 is a potent inhibitor of infections in tissue cul-
ture. Peptides corresponding to equivalent regions of other retroviruses
as well as several paramyxoviruses function similarly (Earp et al. 2003;
Russell et al. 2001). Notably, all of the viruses that have been shown to
be highly susceptible to "C-helix" peptide inhibitors function at neutral
pH, at least up to the lipid interacting stage of virus-cell fusion (Earp et
al. 2003). Peptides corresponding to the N-terminal helices of HIV Env
and the SV5 F protein also inhibit fusion, although with lower potency
(Lu et al. 1995; Russell et al. 2001). The mechanism of inhibition by
N-terminal peptides is still under consideration (He et al. 2003). In the
case of the SV5 F, the N-peptide appears to target an earlier intermedi-
ate than the C-peptide (Russell et al. 2001). Other strategies are being
considered to stabilize the prehairpin intermediate (Fig. 3, Step 1) and
thereby prevent six-helix bundle formation. One strategy is the devel-
opment of antibodies that recognize the prehairpin intermediate
(Golding et al. 2002). Another, exemplified in three studies, is the devel-
opment of small molecules that prevent six-helix bundle formation
(Debnath et al. 1999; Eckert et al. 1999; Ferrer et al. 1999). All three
studies targeted a hydrophobic pocket in the groove of the central
coiled-coil of HIV gp41 that is important for interaction with the C-ter-
minal helix in the post-fusion form. In the first approach, two organic
compounds were identified from a screening effort conducted in
conjunction with molecular docking, a method to identify small mole-
cules that fit in a target site (Debnath et al. 1999). The second approach
replaced three residues of the C-terminal helix that bind to the hydro-
phobic pocket with organic moieties, generated by combinatorial chem-
istry (Ferrer et al. 1999). The third approach used a mirror image phage
display library to identify small, D-amino acid containing peptides that
bind to the pocket (Eckert et al. 1999). Although none of the small mo-
lecules identified to date is as potent as T-20, the precedent has been set
for attaining this goal.

The Many Mechanisms of Viral Membrane Fusion Proteins 51

8.2

Inhibition of Other Steps in Fusion

An effort to block the fusion activity of influenza was based on the idea
that maintaining HA in its native metastable state should prevent fusion
and infection. The first trial targeted a site in X:31 HA that includes part
of the fusion peptide. With the use of an antibody-based assay to
monitor fusion peptide exposure, a compound, tert-butylhydroquinone
(TBHQ), that prevents the first stages of the HA conformational change
and inhibits infectivity was discovered (Bodian et al. 1993). A follow-up
study, targeting a site near the B-loop in HA, yielded additional in-
hibitors. Whereas some functioned like TBHQ, a second class was iden-
tified that appeared to push HA to an inactive state (Hoffman et al.
1997). A random screen against an HI influenza virus identified an in-
hibitor that appears to function similarly to TBHQ. In the latter case,
the binding site for the inhibitor was mapped to the vicinity of the fu-
sion peptide (Cianci et al. 1999). Other small molecules that inhibit con-
formational changes in HA have been identified (Staschke et al. 1998).
To date, none of the HA inhibitors has blocked all HA subtypes and
none has an IC 50 value in the submicromolar range. It is not yet clear
whether the latter limitation represents a fundamental difficulty in in-
hibiting viral fusion proteins that function at low pH.

In addition to the small molecule approaches described above, anti-
bodies that prevent fusion-inducing changes in viral glycoproteins have
been described. The first example was an antibody that prevents low-
pH-induced fusion of West Nile virus with model liposomes (Gollins
and Porterfield 1986). Recently, a Fab fragment that binds to two HA1
monomers was shown to prevent an early conformational change in in-
fluenza HA (Barbey-Martin et al. 2002), separation of the globular head
domains (Fig. 2B). As described above, antibodies have been developed
that likely block six-helix bundle formation in the case of HIV Env
(Golding et al. 2002).

Perspectives

The goal of this review was to give the reader an appreciation for the di-
versity of viral fusion mechanisms while recognizing their common un-
derlying principles. We also summarized what is known about the lipid
dynamics and lipid structures involved in fusion, and we also briefly

L.J. Earp et al.

overviewed recent developments in targeting viral fusion as an antiviral
strategy. There is clearly much more we need to know about viral fusion
proteins, viral fusion reactions, and the design of antifusion agents.

We end this review by enumerating some pressing issues and ques-
tions that remain about viral fusion. A major goal is to determine high-
resolution structures for the complete ectodomains of the metastable
trimers of class I viral fusion proteins in addition to the influenza HA.
Structures of a complete paramyxovirus F and a complete retroviral Env
ectodomain will be highly informative because we currently lack a de-
tailed molecular description of how a receptor activates any viral fusion
protein at neutral pH. A second goal will be to further delineate the
mechanisms of class II viral fusion proteins. Do the transitions to their
recently described low pH forms (Bressanelli et al. 2004; Gibbons et al.
2004; Modis et al. 2004) mediate hemifusion or fusion pore opening?
What about the mechanisms of the as yet unclassified viral fusion pro-
teins? These include viruses such as rhabdoviruses (e.g., VSV) that need
only one protein to promote fusion, as well as more complicated viruses
such as herpesviruses and poxviruses that require multiple viral glyco-
proteins.

The ensuing years should also bring a more complete understanding
of how viral fusion proteins interact with target membrane bilayers.
Class II fusion proteins insert their internal fusion peptides into target
membranes as loops (Bressanelli et al. 2004; Gibbons et al. 2004; Modis
et al. 2004). It has been predicted that the internal fusion proteins of the
class I fusion proteins from Ebola and avian retroviruses form disulfide-
bonded loop structures (Weisenhorn et al. 1998), and mutagenesis work
has supported this prediction (Delos et al. 2000; Delos and White 2000;
Jeffers et al. 2002). It remains to be seen, from high resolution structural
studies, whether all internal fusion peptides, be they from class I, class
II, or other classes of fusion proteins, interact with target bilayers as
(disulfide bond) stabilized loops. Finally, we expect that there will be
major developments in furthering the concept of targeting fusion as a
weapon against pathogenic enveloped viruses. Particular emphasis will
likely be on the development of small molecule inhibitors through the
use of combinatorial chemistry in conjunction with high-throughput
screens. It will be interesting to learn whether small molecule fusion in-
hibitors can be identified that block the entry of viruses that fuse in en-
dosomes in response to low pH. This is a challenge for low-pH-activated
class I fusion proteins such as influenza HA as well as for all known class
II fusion proteins. Stay tuned. There are likely to be exciting develop-

The Many Mechanisms of Viral Membrane Fusion Proteins 53

ments in our understanding of viral fusion mechanisms as well as in the
development of antifusion antivirals in the years ahead.

Acknowledgements Work in the authors' laboratory was supported by the NIH
(AI22470). We thank Dr. Margaret Kielian for helpful comments on the manuscript.

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CTMI (2004) 285:67-108
© Springer- Verlag 2004

The Role of the Cytoskeleton During Viral Infection

K. Dohner • B. Sodeik (El)

Department of Virology, OE 5320, Hannover Medical School,
Carl-Neuberg-Str. 1, 30625 Hannover, Germany
Sodeik.Beate@MH-HANNOVER.DE

1 Introduction

2 The Cytoskeleton . . .

2.1 Intermediate Filaments

2.2 Actin Filaments ....

2.3 Microtubules

3 Molecular Motors. . .

3.1 Myosins

3.2 Kinesins

3.3 Cytoplasmic Dynein .

70
71
71

74
74
75
76

3.3.1 Binding Partners of Dynein Light Chains 77

4 Experimental Approaches

4.1 The Tool Kit

4.2 Video Microscopy 79

4.3 Virological Assays, Cytoskeletal Cooperation, and Viral Modifications . 80

5 Neurotropic Herpesvirus and Cytoplasmic Transport 82

5.1 Microtubule Transport During Entry 82

5.2 Microtubule Transport During Egress 84

5.3 Biochemical Analysis of Viral Capsid Transport 86

5.4 Interaction of Herpesvirus with Cytoskeletal Proteins 87

6 Retrovirus Entry and Budding

7 Adenovirus Entry and Microtubules 90

8 Parvovirus Entry and Microtubules 91

9 Poxviruses — Multiple Cargos for Microtubules and Actin Tails 92

9.1 Cytosolic Vaccinia Virus Cores 92

9.2 Particle Transport During Virus Egress 93

10 Actin Remodelling During Baculovirus Infection 94

1 1 Perspectives

References

K. Dohner • B. Sodeik

Abstract Upon infection, virions or subviral nucleoprotein complexes are transport-
ed from the cell surface to the site of viral transcription and replication. During viral
egress, particles containing viral proteins and nucleic acids again move from the site
of their synthesis to that of virus assembly and further to the plasma membrane. Be-
cause free diffusion of molecules larger than 500 kDa is restricted in the cytoplasm,
viruses as well as cellular organelles employ active, energy-consuming enzymes for
directed transport. This is particularly evident in the case of neurotropic viruses that
travel long distances in the axon during retrograde or anterograde transport. Viruses
use two strategies for intracellular transport: Viral components either hijack the cy-
toplasmic membrane traffic or they interact directly with the cytoskeletal transport
machinery. In this review we describe how viruses — particularly members of the
Herpesviridae, Adenoviridae, Parvoviridae, Poxviridae, and Baculoviridae — make
use of the microtubule and the actin cytoskeleton. Analysing the underlying princi-
ples of viral cytosolic transport will be helpful in the design of viral vectors to be
used in research as well as human gene therapy, and in the identification of new an-
tiviral target molecules.

Introduction

As obligate intracellular parasites, viruses use and manipulate the cell's
machinery for membrane trafficking, transcription, splicing, nuclear
pore transport and protein synthesis. In fact, these cellular processes
were elucidated to a large extent by studying viral systems. Here, we dis-
cuss the molecular interactions of viruses with the host cytoskeleton
and the mechanisms of viral cytoplasmic transport.

Any viral life cycle can be divided into the following phases: (1) ad-
sorption to the cell, (2) penetration of the plasma or endosome mem-
brane, (3) genome uncoating and release from a viral nucleoprotein
complex or capsid, (4) early viral protein synthesis, (5) genome replica-
tion, (6) late viral protein synthesis, (7) virus assembly/maturation and
finally (8) viral release or egress. We define phases (l)-(3), up to the
stage at which the viral genome has been unpacked for transcription
and replication, collectively as "viral cell entry".

Virions, subviral particles or nucleoprotein complexes are transport-
ed during cell entry from the cell surface to the site of viral transcription
and replication, as well as from the site of synthesis to that of virus as-
sembly and back to the plasma membrane for virus egress (for recent
reviews see Cudmore et al. 1997; Sodeik 2000; Ploubidou and Way 2001;
Smith and Enquist 2002). Because free diffusion of molecules larger than
500 kDa is restricted in the cytoplasm compared to dilute solutions,
viruses as well as cellular organelles and chromosomes depend on active

The Role of the Cytoskeleton During Viral Infection

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K. Dohner • B. Sodeik

mechanisms for directed transport (Luby-Phelps 2000). This is particu-
larly evident in the case of neurotropic viruses that travel long distances
in the axon during retrograde or anterograde transport (Smith and
Enquist 2002). For example, it would take a herpes virus capsid 231 years
to diffuse 10 mm in the axonal cytoplasm (Sodeik 2000).

Viruses use two alternative strategies for intracellular transport. One
is to hijack cytoplasmic membrane traffic. During cell entry many virus-
es pass through the endocytic pathway to the cell centre (see the chapter
by Sieczkarski and Whittaker, this volume). After budding, virions travel
inside vesicles derived from the endoplasmic reticulum and the Golgi
apparatus to the plasma membrane for viral egress (see the chapter by
Maggioni and Braakman, this volume). Alternatively, viral components
interact directly with the cytoskeletal transport machinery. It is helpful
to distinguish between cytoplasmic and cytosolic transport. The first is
a general term referring to both transport inside a membrane and cyto-
solic transport. The latter involves direct interactions between cytosolic
viral components and the cytoskeleton (Sodeik 2000).

We begin with an overview of the organisation and function of the cy-
toskeleton and then discuss selected viruses whose cytoplasmic trans-
port is either well characterised in molecular terms, such as herpesvirus
and adenovirus, or mechanistically unique, as for vaccinia virus and
baculovirus (Table 1).

The Cytoskeleton

The host cytoskeleton forms a three-dimensional network which is regu-
lated by many accessory proteins and defines the cell's shape as well as
its internal organisation. It mediates physical robustness, cell-cell con-
tact, cell crawling, cell division, organelle or RNA transport during inter-
phase and chromosome movement during mitosis or meiosis. Cytoskel-
etal filaments are assembled by non-covalent binding from protein sub-
units: Monomeric actin polymerises into microfilaments with a diameter
of 5-6 nm, dimeric tubulin assembles hollow tubes of 25 nm and pro-
teins such as keratin or vimentin form 10-nm intermediate filaments.

The Role of the Cytoskeleton During Viral Infection 71

2.1

Intermediate Filaments

The intermediate filament proteins show a characteristic tripartite do-
main organization. N- and C-terminal globular head and tail domains of
varying size and sequence flank a centrally located rod domain which
mediates lateral interactions during assembly (Coulombe et al. 2001).
The rope-like intermediate filaments span the entire cytosol and provide
mechanical strength against shear forces.

Intermediate filaments do not seem to play any role in intracellular
transport because they have no polarity and no motor proteins have
been identified which use them as tracks.

During the late phase of many viral infections, intermediate filaments
are rearranged or disassembled by viral proteases (Chen et al. 1993;
Ferreira et al. 1994; Luftig and Lupo 1994; Brunet et al. 2000). If the in-
termediate filaments are disassembled, microinjected beads can diffuse
more freely (Luby-Phelps 2000). One could assume that the same holds
true for viral particles, and that viral modification of the intermediate
filaments facilitates egress.

The formation of aggresomes also leads to the reorganisation of inter-
mediate filaments into a perinuclear cage in a cellular attempt to seques-
ter misfolded and unassembled protein subunits (Garcia-Mata et al.
1999; Kopito 2000). African Swine Fever virus might actually exploit this
pathway in order to concentrate viral structural proteins for assembly in
a viral factory (Heath et al. 2001).

2.2

Actin Filaments

The most abundant protein of many cells, actin, exists in monomeric
form as globular actin or G-actin and in filamentous form called F-actin
or microfilaments. Polymerisation proceeds by the reversible endwise
addition of actin subunits that stimulates hydrolysis of bound ATP. Each
filament is polarised, and its ends have distinct biochemical properties.
The fast-growing end is called the barbed or plus-end, and the slow-
growing end the pointed or minus-end (Welch and Mullins 2002). In
cells, polymerisation occurs primarily at the plus-end and must be tight-
ly regulated, because the turnover of actin subunits in vivo is 100-200
times faster than with pure actin (Zigmond 1993).

Actin filaments are flexible structures which are organised into a vari-
ety of linear bundles and arrays, two-dimensional networks or three-

K. Dohner • B. Sodeik

dimensional gels. Numerous G- and F-actin binding proteins regulate
the monomer pool, the formation of filaments and higher-order net-
works as well as filament depolymerisation for monomer recycling. For
example, binding of the Arp2/3 complex to the lateral side of actin fila-
ments leads to a dendritic pattern of actin filament nucleation (Welch
and Mullins 2002).

Besides actin filaments dispersed throughout the entire cell, all cells
contain a cortical network of actin filaments, generally oriented with
their plus-ends facing towards the plasma membrane. This actin cortex
is the key player in various cell motility processes which are based on
filament assembly and the action of myosins (Welch and Mullins 2002;
Kieke and Titus 2003).

However, the actin cortex beneath the plasma membrane can also be
an obstacle for virus entry or budding (Marsh and Bron 1997). This may
be the reason why many viruses enter cells via endosomes which easily
traverse the actin cortex (see the chapter by Sieczkarski and Whittaker,
this volume). Certain viruses might even fuse with either the plasma
membrane or an endosome depending on the cell type they are infecting
(Miller and Hutt-Fletcher 1992; Nicola et al. 2003).

The cortical actin might also be a barrier for virus assembly at the
plasma membrane. The cortical actin might be locally depolymerised,
or the force of actin polymerisation could be used to drive the budding
process (Cudmore et al. 1997; Garoff et al. 1998). Recent evidence sug-
gests that a shell of cortical actin also surrounds many membrane or-
ganelles. Thus viruses could pick up actin and actin-binding proteins
during budding as has been shown for human immunodeficiency virus
(HIV) (Ott et al. 1996; Liu et al. 1999; Wilk et al. 1999).

Regulated binding to cytosolic actin filaments can control the subcel-
lular localisation of a protein; an example is the nucleoprotein of influ-
enza virus (Digard et al. 1999). Despite many reports on nuclear actin
only recent evidence supports a role of nuclear actin in chromatin re-
modelling, splicing, nuclear import and export (reviewed in Rando et al.
2000; Pederson and Aebi 2002). Many viruses use actin during replica-
tion, but with the exception of nucleocapsid assembly of some bac-
uloviruses (Kasman and Volkman 2000) and the export of unspliced
genomic HIV-1 RNAs (Kimura et al. 2000; Hofmann et al. 2001), these
activities all occur within the cytoplasm.

The Role of the Cytoskeleton During Viral Infection 73

2.3

Microtubules

Microtubules (MTs) are long, hollow cylinders assembled from het-
erodimers of a- and /^-tubulin and MT-associated proteins (MAPs). The
head-to- tail association of tubulin into 13 protofilaments leads to the for-
mation of MTs, which are further stabilised by lateral interactions between
adjacent protofilaments. MTs are, like actin filaments, polar structures
with a dynamic fast-growing plus-end and a less dynamic minus-end
(Desai and Mitchison 1997; Downing 2000). The overall subcellular MT
organisation is highly polarised. MT dynamics vary considerably between
different regions of a cell, during the cell cycle and throughout differentia-
tion. Thus MT turnover is tightly controlled in space and over time.

The plus-ends of MTs are the primary sites of MT growth and short-
ening, resulting in a phenomenon called dynamic instability (Mitchison
and Kirschner 1984). After subunit addition at the end, the GTP bound
to /^-tubulin is slowly hydrolysed. The consecutive loss of this GTP- tubu-
lin cap leads to catastrophic MT depolymerisation. GTP-tubulin is more
likely to be added to a GTP-tubulin cap than to a MT rapidly depoly-
merising. Plus-end binding proteins such as CLIP- 170, EB1 or APC sta-
bilise a region of about 1 jum at the plus-end of MTs and thus enable
them to interact with the plasma membrane, organelles or the kineto-
chores of chromosomes. This search-and-capture mechanism prevents
depolymerisation, and the MT stabilisation can even reorient the MTOC
and thus the entire MT network (Howard and Hyman 2003).

In most animal cells, minus-ends are attached to a MT-organising
centre (MTOC) called a centrosome and typically located close to the cell
nucleus whereas the plus-ends are pointing towards the cell periphery
and plasma membrane. However, several cell types contain a substantial
number of non-centrosomal MTs (Keating and Borisy 1999). Even at
steady state — when a population of MTs has reached constant mass —
individual MTs may be polymerising or depolymerising, and such
dynamics modulate intracellular transport (Keating and Borisy 1999;
Giannakakou et al. 2002). Polarised epithelial cells are characterised by
an apical and a basal MT web and longitudinally arranged MTs which
point with their minus-ends to the apical and with their plus-ends to the
basal membrane (Topp et al. 1996). Neuronal MTs in axons are also lon-
gitudinally arranged with their minus-ends pointing to the soma and
their plus-ends to the axon terminal, whereas most dendrites have MTs
of mixed polarity (Goldstein and Yang 2000; Hirokawa and Takemura
2003).

K. Dohner • B. Sodeik

Many MAPs such as tau contain a basic domain which interacts with
MTs via the acidic C-terminal domains of tubulin which are exposed on
the MT surface (Downing 2000). Also, viral proteins with a basic domain
such as many RNA- or DNA-binding proteins have the potential to inter-
act with the MT surface (Elliott and O'Hare 1997; Ploubidou et al. 2000;
Mallardoetal. 2001).

MTs are involved in long-distance transport as highlighted by the
transport requirements through neuronal dendrites and axons. More-
over, the reversible association to MTs and motors regulates the subcel-
lular localisation of important signalling proteins and transcription fac-
tors (Ziegelbauer et al. 2001; Giannakakou et al. 2002; Schnapp 2003).

Molecular Motors

Many proteins interact with tubulin and actin to regulate their structural
organisation. A subset of those, the motor proteins, use conformational
changes powered by ATP hydrolysis to transport cargo along the fila-
ments. If motors are fixed on a surface such as a membrane, they move
the filaments relative to that surface. If instead the filament is fixed, mo-
tors transport cargo along that filament.

Myosins mediate translocation along actin filaments, whereas dynein
and kinesins move on MTs. It has been suggested that in animal cells
long-range transport occurs along MTs and short-range transport along
actin filaments (reviewed in Brown 1999; Verhey 2003). A viral particle
could be transported either by directly recruiting a motor or by attach-
ing to a cargo of a given motor. Moreover, polymerising actin filaments
can push around different cargo such as endocytic vesicles, intracellular
bacteria or vaccinia virus (Frischknecht and Way 2001).

3.1
Myosins

All myosins share a common N-terminal motor domain of about
500 amino acids that binds to actin filaments (www.mrc-lmb.cam.ac.uk/
myosin/myosin.html). A flexible neck region connects the motor domain
to a tail region of variable size which determines the specific function of
each myosin and the specificity of cargo binding. Myosins have been
grouped into 18 classes according to homologous sequences in the tail
domain (Kieke and Titus 2003).

The Role of the Cytoskeleton During Viral Infection 75

Most myosins walk towards the plus-ends of actin filaments, with the
exception of myosin VI and possibly myosin IXb, which are minus-end-
directed motors (Kieke and Titus 2003). The myosin lis are often called
'conventional' myosins and include the best-studied example of skeletal
muscle myosin. Cytoplasmic myosin II consists of two heavy chains and
two pairs of light chains. Myosin II generates contractile forces during
cytokinesis, maintains cortical tension and is crucial for cell motility.
Myosin I, V and VI are involved in membrane traffic; moreover, myosin
I plays a role in cell motility and signal transduction (Kieke and Titus
2003).

3.2

Kinesins

Kinesins transport membranes, cytoskeletal filaments, viral particles,
mRNAs and proteins along MTs and are engaged in organising the mi-
totic spindle as well as chromosome translocation (Hirokawa and Take-
mura 2003). The defining criterion for a kinesin is a MT-binding motor
domain of 320 amino acids, which is folded into a core with an overall
structure similar to myosins and G proteins (Schliwa and Woehlke 2003;
www.proweb.org/kinesin//index.html). The motor domain is linked to
structural and regulatory domains, which attach to other cofactors,
adaptor proteins or interaction modules (Karcher et al. 2002; Schnapp
2003). The kinesins are sorted into three groups according to the posi-
tion of the motor domain at the N-terminus, an internal position, or the
C-terminus, hence the names Kin-N, Kin-I and Kin-C kinesins, and
based on homology into 14 classes (Hirokawa and Takemura 2003).

N-type kinesins, which include conventional kinesin (kinesin- 1,
KIF5), move towards MT plus-ends. Kinesin- 1 is a heterodimer of two
heavy chains (KHC) that each contain a motor domain, an a-helical
stalk responsible for dimerization and a C-terminal tail domain that
binds two light chains (KLC). The KLCs can dock onto adaptors or cargo
receptors and link kinesin to the cargo (Hirokawa and Takemura 2003).
Kinesin- 1 transports various cargos such as axonal vesicles, mitochon-
dria, lysosomes, endocytic vesicles, tubulin oligomers, intermediate fila-
ment proteins or mRNA complexes (Hirokawa and Takemura 2003). Ki-
nesin- 1 is also responsible for the transport of vaccinia virus (VV) to
the plasma membrane (Rietdorf et al. 2001).

The respective cargos of the N-type kinesins KIF1A and KIF1B are
synaptic vesicles and mitochondria (Nangaku et al. 1994; Okada et al.
1995). The N-type heterotrimeric kinesin with the KIF3 heavy chains

K. Dohner • B. Sodeik

mediates axonal transport of fodrin-containing vesicles, and in epithelial
cells KIF3 interacts with APC, a MT plus-end binding protein (Takeda et
al. 2000; Jimbo et al. 2002). Kin-C kinesins move towards the MT minus-
ends (Hirokawa and Takemura 2003). Most C-type kinesins act during
mitosis, but some are implicated in membrane transport in neuronal
dendrites and polarised epithelial cells (Hanlon et al. 1997; Saito et al.
1997; Noda et al. 2001; Xu et al. 2002). Kin-I kinesins most likely do not
work as motors but destabilise MTs (Desai et al. 1999; Hunter et al.
2003).

3.3

Cytoplasmic Dynein

Axonemal and cytoplasmic dyneins transport cargo towards MTs mi-
nus-ends. Axonemal dyneins provide the driving force for the beating
movement of cilia and flagella (King 2003). Cytoplasmic dynein plays an
essential role during mitosis and is responsible for the perinuclear local-
isation of several organelles around the MTOC as well as retrograde or-
ganelle transport in axons (Karki and Holzbaur 1999). Cytoplasmic dy-
nein is also required for the transport of non-membranous cargo such
as NuMA, aggresomes, viral capsids, neurofilaments and pericentrin
particles (Merdes and Cleveland 1997; Garcia-Mata et al. 1999; Suoma-
lainen et al. 1999; Shah et al. 2000; Sharp et al. 2000; Young et al. 2000;
Dohner et al. 2002). It is even involved in the transport of mRNAs and
proteins regulating transcription (Galigniana et al. 2001; Giannakakou et
al. 2002; Tekotte and Davis 2002).

Cytoplasmic dynein is a 20 S protein complex in the shape of a Y and
consists of two dynein heavy chains (DHCs; 530 kDa), two intermediate
chains (DICs; 70-80 kDa), four light intermediate chains (DLICs; 50-
60 kDa), and three families of light chains (DLCs, 7-14 kDa, see below;
Karki and Holzbaur 1999; King 2003). DHCs are members of the AAA
family of ATPases and mediate MT binding, ATP hydrolysis and force
generation (King 2000). The DICs belong to the WD-repeat protein fam-
ily and are located at the base of the dynein motor. DLICs, DICs and
DLCs have been implicated in motor regulation and cargo binding (King
2003). The subunits differ in their subcellular localisation and tissue ex-
pression, suggesting that cytoplasmic dynein exists in many distinct iso-
forms with unique subunit composition (Nurminsky et al. 1998; Tai et
al. 2001).

For most, if not all, minus-end-directed transport processes cytoplas-
mic dynein requires a cofactor called dynactin (Karki and Holzbaur

The Role of the Cytoskeleton During Viral Infection 11

1999). Dynactin is another large 20 S protein complex consisting of 11
different subunits (Holleran et al. 1998; Eckley et al. 1999). The pl50 Glued
subunit of dynactin binds directly to the DICs. Interestingly, dynactin
was recently reported to also play a role in melanosome transport catal-
ysed by heterotrimeric kinesin (Deacon et al. 2003).

3.3.1

Binding Partners of Dynein Light Chains

Many DLC binding partners have been identified in yeast two-hybrid
screens, sometimes supported by GST-pull down or co-immunoprecipi-
tation assays after transient overexpression of the proteins. However, it
has often been difficult to confirm these interactions with endogenous
proteins under physiological conditions, and to prove that they lead to
the recruitment of a motor. Based on sequence homology, DLCs have
been grouped into the LC7/Roadblock, LC8/PIN and Tctex families (King
2003).

Members of the LC7 family bind to DICs and play a role in intracellu-
lar transport and mitosis in Drosophila (Bowman et al. 1999; Susalka et
al. 2002). A mammalian member, mLC7, interacts with transforming
growth factor-/? (Tang et al. 2002).

DLCs of the LC8/PIN family bind to neuronal nitric oxide synthase
and are therefore also called PIN for 'protein inhibitor of nitric oxide
synthase' (Jaffrey and Snyder 1996). Further interaction partners are
I/cBa, Bim of the Bcl-2 family, postsynaptic density- 9 5/guanylate kinase
domain-associated protein, nuclear respiratory factor, the erect wing
gene product in Drosophila, swallow protein, DIC and, interestingly, the
3 / -UTR of parathyroid hormone RNA (King 2003). LC8/PIN is also a
subunit of the actin motor myosin V (Espindola et al. 2000). The inter-
action often involves a conserved K/R-XTQT or G-I/V-QVD sequence
motif in the DLC binding partner (Lo et al. 2001; Rodriguez-Crespo et
al. 2001; Martinez-Moreno et al. 2003).

LC8/PIN also interacts with the phosphoprotein of rabies and Mokola
virus (Raux et al. 2000; Jacob et al. 2000). Rabies virus spreads via neu-
rons and travels long distances from the site of entry to the neuronal cell
body. Axonal transport of rabies virus requires intact MTs, but it is un-
clear whether vesicles containing virions or cytosolic capsids are trans-
ported (Ceccaldi et al. 1989; Tsiang et al. 1991). Rabies viruses deleted
for the LC8 binding motif no longer incorporate LC8, but pathogenesis
is only impaired in already attenuated strains, and only in very young
mice (Mebatsion 2001; Poisson et al. 2001). LC8/PIN also binds to p54 of

K. Dohner • B. Sodeik

African swine fever virus, whose infection is inhibited if dynein is
blocked (Alonso et al. 2001; Heath et al. 2001). Yeast two-hybrid screens
revealed an interaction between HIV integrase and DYN2, a putative
LC8-like yeast dynein light chain of 92 amino acids (Richard de Soultrait
et al. 2002). Several additional potential viral interaction partners for
LC8/PIN such as HSV1 helicase, adenovirus protease, vaccinia virus
( VV) polymerase and, surprisingly, the extracellular domain of respira-
tory syncytial virus attachment protein have been identified by screen-
ing libraries of overlapping dodecapeptides in ligand blots (Martinez-
Moreno et al. 2003).

DLCs of the Tctex class bind to Doc2, p59 fyn kinase, rhodopsin, FIP1,
Trk receptors, DIC, the cytosolic tail of CD5 and CD 155, often via the
binding motif K/R-K/R-XX-K/R (King 2003). CD155 is the receptor for
poliovirus that invades the CNS by haematogenous or neural spread
(Mueller et al. 2002). After binding to the receptor the poliovirus ge-
nome is released into the cytosol through a membrane pore. It is unclear
whether pore formation occurs already at the plasma membrane or after
delivery to an endosome (Hogle 2002). If axonal transport occurs inside
an endocytic membrane, the binding of CD 155 to Tctex- 1 and thus
possibly dynein could mediate retrograde transport of virus -containing
vesicles to the cell body of the motor neuron (Mueller et al. 2002).

Experimental Approaches

4.1

The Tool Kit

There are several reversible pharmacological drugs that target cytoskele-
tal proteins. The response of different cell types to these drugs varies
greatly, and therefore each inhibitor is tested initially with different con-
centrations and incubation times (Jordan and Wilson 1999).

Cytochalasin and latrunculin depolymerise actin filaments, whereas
jasplakinolide promotes actin polymerisation (Cooper 1987; Bubb et al.
1994; Ayscough 1998). Colchicine, colcemide, vincristine, vinblastine
and nocodazole (podophyllotoxin) depolymerise MTs, whereas taxol
(paclitaxel) stabilises the MT network (Jordan and Wilson 1999). Be-
cause all these are not competitive inhibitors but interfere with the dy-
namic equilibrium between subunits and filaments, the cells need to be
treated for quite some time, until an effect on the steady-state distribu-

The Role of the Cytoskeleton During Viral Infection 79

tion of the cytoskeleton is manifested (Cooper 1987; Jordan and Wilson
1999).

Butanedione monoxime (BDM) inhibits ATP hydrolysis of myosin II,
V, VI and possibly I (Herrmann et al. 1992; Cramer and Mitchison
1995). However, higher concentrations may also target other ATPases
(Schlichter et al. 1992; Mojon et al. 1993; Phillips and Altschuld 1996).
KT5926, wortmannin and ML-7 block the activity of myosin light chain
kinase, and thus the only known downstream target, the non-muscle
myosin II (Nakanishi et al. 1990; Nakanishi et al. 1992; Ruchhoeft and
Harris 1997). Adociasulfate blocks kinesins (Sakowicz et al. 1998), and
dyneins are inhibited by erythro-9- [3- (2-hydroxynonyl)] adenine that
also affects adenosine deaminase and cGMP-stimulated phosphodiester-
ase (Penningroth 1986; Mery et al. 1995).

Microinjecting function-blocking antibodies, adding anti-sense or
small interfering RNAs or overexpressing a dominant-negative protein
can inhibit many motors. For example, excess dynamitin blocks dyn-
actin and overexpression of the cargo-binding domain of KLC inhibits
conventional kinesin (Echeverri et al. 1996; Burkhardt et al. 1997; Valetti
et al. 1999; Rietdorf et al. 2001).

Testing many inhibitors aiming at alternative targets by different mo-
lecular mechanisms provides a guide for analysing the role of the cyto-
skeleton in virus infection. To determine which viral proteins are in-
volved in cytoplasmic transport, viral mutants with defined genotypes
have been generated.

4.2

Video Microscopy

Intracellular movement of host and viral particles is best analysed by vi-
deo or digital time-lapse microscopy (Lippincott- Schwartz et al. 2000;
Zhang et al. 2002). This technique adds another dimension to the analy-
sis of fixed cells, because not only the steady-state distribution of viral
components but also the dynamics of their transport is analysed. Be-
cause of their small size and limited contrast, viral particles with the ex-
ception of vaccinia virus need to be tagged with a fluorescent molecule
for tracking in live cells.

Direct chemical coupling to small fluorescent dyes was used to ana-
lyse cytoplasmic transport of non-enveloped viruses (Georgi et al. 1990;
Leopold et al. 1998; Suomalainen et al. 1999). Because many viruses un-
dergo substantial disassembly steps during entry (Greber et al. 1993),

K. Dohner • B. Sodeik

one needs to identify the protein to which the dye has been attached
and, if possible, analyse trafficking of differently tagged viral structures.

To analyse the infection of enveloped viruses, the fluorescent tag must
be linked to a structure which after membrane fusion remains associat-
ed with the genome during its cytosolic passage. Green fluorescent pro-
tein (GFP)-tagged capsid or core proteins have been used to track pseu-
dorabies virus and HIV (Smith et al. 2001; McDonald et al. 2002). Be-
cause all viral particles can be taken up by endocytosis, even if that is
not their physiological entry port (see the chapter by Sieczkarski and
Whittaker, this volume), experiments using fluorescently labelled virions
require care to distinguish cytosolic particles from those in endosomes
(Suomalainen et al. 1999; McDonald et al. 2002; Sodeik 2002). Likewise,
when analysing viral egress, one needs to distinguish GFP-tagged virions
from GFP-fusion protein in the biosynthetic pathway or in the cytosol
(Rietdorf et al. 2001). The recent improvements in the design of fluores-
cent proteins allow dual-colour imaging of different viral particles in live
cells (Zhang et al. 2002).

Attaching multiple fluorescent molecules to a virus or subviral parti-
cle could alter virus assembly and subsequently virus entry. Fluorescent
dyes are usually quite hydrophobic, and GFP is a protein of 27 kDa re-
sulting in a rather large mutation of any viral protein. Therefore, it is
crucial to test with several assays that a GFP-tagged virus behaves simi-
larly to the unlabelled virus. Among the physiological cargo of dynein
are aggregated protein complexes that are packed into aggresomes at the
MTOC (Garcia-Mata et al. 1999; Kopito 2000). Thus the cells might send
many non-functional viral proteins along MTs to the MTOC for refold-
ing or final degradation (Sodeik 2002).

Many of these issues can now be overcome by single-molecule imag-
ing techniques (Seisenberger et al. 2001). However, they require spe-
cialised equipment to visualise the emission with a low signal-to-noise
ratio. Most results so far have been obtained with a high ratio of dye to
particle. With the use of fluorescent proteins with improved excitation
and emission spectra and digital cameras with increasing sensitivity,
movies of viral cytoplasmic transport at physiological conditions can
now be recorded.

4.3

Virological Assays, Cytoskeletal Cooperation, and Viral Modifications

Besides suitable tools to analyse the cytoskeleton, many experiments
have been developed to study individual phases of a viral life cycle. In

The Role of the Cytoskeleton During Viral Infection 81

addition to blocking cytoplasmic transport, the inhibitors used could
also influence viral infection in other ways.

For example, because a cell's membrane traffic relies heavily on actin
and MT transport, important host factors, such as viral receptors, could
mis-localise, thus resulting in a reduced or increased virus binding, inter-
nalisation or other changes in the viral life cycle (Dohner et al. 2002). If
the readout for cytoplasmic transport during virus entry is nuclear viral
gene expression, a potential role of nuclear actin or myosin in viral tran-
scription should be considered (Pederson and Aebi 2002). The host cyto-
skeleton can also directly influence viral transcription as shown for
paramyxoviruses or reverse transcription and viral budding as in the case
of HIV (Sasaki et al. 1995; Bukrinskaya et al. 1998; Gupta et al. 1998).

Moreover, the three types of cytoskeletal filaments are interdependent
entities which operate together in cells (Fuchs and Yang 1999; Goode et
al. 2000). The last years have shown a close interaction between MT
plus-ends and the actin cortex, and several proteins provide physical
links between different filament types (Coulombe et al. 2000; Allan and
Nathke 2001; Leung et al. 2002). Thus a prolonged incubation with any
of the inhibitors could lead to pleiotropic effects. For example, targeting
MTs with depolymerising drugs or with antibodies to stable MTs also re-
sults in the collapse of intermediate filaments to a perinuclear region
(Gurland and Gundersen 1995).

Many cells contract, and in the extreme case completely round up, if
actin filaments are disassembled, because their substrate adhesion via
focal contacts is weakened (Ayscough 1998). This reduces the transport
distances, for example from the plasma membrane to the nucleus, and
cargo could reach a particular binding site such as the nuclear pore in
the absence of the appropriate filament system by diffusion rather than
by active transport as under physiological conditions. Moreover, al-
though cytoskeletal filaments provide tracks for cytoplasmic viral trans-
port, they also restrict the space that is open for translocation (Luby-
Phelps 2000). Thus depolymerising cytoskeletal filaments removes both
transport trails and steric barriers.

For these reasons, it is sometimes difficult to determine the precise
functional role of the cytoskeleton during virus infection. In addition,
late in infection the cytoskeletal filaments are often rearranged, compli-
cating the analysis of their role during assembly and egress (Avitabile et
al. 1995; Brunet et al. 2000; Dreschers et al. 2001). However, by taking
these caveats and limitations into consideration, we have learnt an amaz-
ing amount about the molecular cross-talk between viruses and the host
cytoskeleton.

K. Dohner • B. Sodeik

Neurotropic Herpesvirus and Cytoplasmic Transport
5.1

Microtubule Transport During Entry

Neurotropic viruses are dependent on efficient transport because they
travel long distances during pathogenesis. Prominent examples are the
alphaherpesviruses such as human herpes simplex virus (HSV). After
initial replication in exposed mucosal epithelia, progeny virions enter lo-
cal nerve endings of peripheral neurons and the capsid without the en-
velope is retrogradely transported to the nucleus located in a peripheral
nerve ganglion. Here, HSV1 establishes a latent infection that on stress
can be reactivated to a lytic infection of the neuron. Newly synthesised
virus is then released from the nerve endings and re-infects the periph-
eral epithelial tissue (Roizman and Knipe 2001; Smith and Enquist
2002). MTs are required in epithelial and neuronal cells as well as
during entry and egress (Kristensson et al. 1986; Topp et al. 1994, 1996;
Avitabile et al. 1995; Sodeik et al. 1997; Miranda-Saksena et al. 2000;
Kotsakis et al. 2001; Mabit et al. 2002).

The fusion of the HSV1 envelope with the plasma membrane releases
into the cytosol a capsid and about 20 different proteins of the tegument,
a protein layer encasing the capsid. The capsid, associated with a subset
of tegument proteins, is transported along MTs to the MTOC, localised
in the cell centre, and further to the nucleus (Kristensson et al. 1986; Ly-
cke et al. 1988; Sodeik et al. 1997; Mabit et al. 2002). The docking of
the capsid at the nuclear pore induces the translocation of the viral ge-
nome into the nucleoplasm (Ojala et al. 2000), where viral replication,
transcription and later during infection capsid assembly take place
(Fig. 1A).

Incoming HSV1 capsids co-localise with cytoplasmic dynein and dy-
nactin in epithelial cells (Sodeik et al. 1997; Dohner et al. 2002). Electron
micrographs of detergent- extracted, infected cells reveal structures re-
sembling the Y-shaped cytoplasmic dynein at the capsid vertices (Sodeik
et al. 1997), where the tegument has a more ordered structure and the
tegument protein VP1-3 might be located (Zhou et al. 1999). Moreover,
inhibiting dynein function by the overexpression of dynamitin reduces
transport of HSV 1 capsids to the nucleus and early viral gene expression
without having any effect on virus binding or internalisation (Dohner et
al. 2002). Whether and how capsids move further from the MTOC to the
nuclear pore complex is unclear (Sodeik 2002).

The Role of the Cytoskeleton During Viral Infection

A- Herpes Simplex Virus

cytoplasm

nucleus

chnrin'dvnaclin

viral DNA

B. HIV

PIC

praviral DMA ah J
u^S-ci-cialcdl pruluin*

C Adenovirus

endosome

viral DNA and
associated proteins

dvnein/tfvnactiTi

microtubule

.*..

200 n in

Fig. 1A-C. Cytosolic transport during virus entry. A After fusion of the envelope of
herpes simplex virus type 1 (HSV1) with the plasma membrane, the capsid (dark
green) and the tegument proteins (green) are released into the cytosol. The capsid
with a subset of tegument proteins associated binds to microtubules (red) and is
transported by dynein and dynactin (blue) to the MT- organising centre where the
MT minus-ends are located. After capsid (light green) binding to the nuclear pore
complex, the viral DNA is injected into the nucleoplasm. B Human immunodeficien-
cy virus type-1 (HIV-1) enters cells by fusion with the plasma membrane. During
passage through the cytosol, the viral RNA genome is reverse transcribed into DNA
in a structure called reverse transcription complex or preintegration complex (PIC;
green). This structure co-localises with microtubules (red) and requires dynein
(blue) for its transport to the nucleus. The proviral DNA is imported into the nucle-
us and integrated into a host chromosome. C Adenovirus of the subgroup C is inter-
nalised by receptor-mediated endocytosis and induces lysis of the endosome (grey)
to access the cytosol. The cytosolic capsid (green) binds to microtubules (red) and is
transported by dynein and dynactin (blue) to the cell centre. During entry, the cap-
sids are stepwise disassembled (indicated by the different shades of green). At the
nuclear pores, the viral DNA and associated proteins are released from the capsid
(light green) and imported into the nucleoplasm

In vivo analysis of the entry into epithelial cells by time-lapse digital
fluorescence microscopy showed that GFP-tagged capsids moved along
MTs towards and away from the nucleus with maximal speeds of
1.1 pm/s (Dohner, Buttner, Wolfstein, Schmidt and Sodeik, in prepara-
tion). Incoming pseudorabies virus, another alphaherpesvirus, moves at
rates averaging 1.3 pm/s. The transport is saltatory and bi-directional,

K. Dohner • B. Sodeik

but in neuronal processes with a retrograde bias towards the cell body
(Smith and Enquist 2002).

Alphaherpesvirus capsids must possess a viral receptor either for dy-
nein or for dynactin, which allows them to engage the host MT system
for efficient transport from the plasma membrane to the host nucleus, a
particularly long distance after infection of neurons via a synapse. In
unpolarised epithelial cells capsids show minus- and plus-ended direct-
ed transport during entry, whereas in cultured neurons the retrograde
transport towards the nucleus predominates.

5.2

Microtubule Transport During Egress

Herpesvirus capsids assemble in the nucleus and after genome packag-
ing acquire a primary envelope by budding at the inner nuclear mem-
brane. The traditional view was that from then on the virus particle re-
mained within the secretory pathway and was released from infected
cells by fusion of a vesicle containing the enveloped virion with the plas-
ma membrane (reviewed in Roizman and Knipe 2001; Mettenleiter
2002). In this scenario, all tegument proteins were added to the capsids
already in the nucleus, and cytoplasmic transport was mediated by the
interaction of membrane vesicles, possibly modified by the addition of
viral proteins, with the host cytoskeleton.

Several studies in the last decade suggested that many if not all her-
pesviruses use an alternative pathway. Accordingly, transiently en-
veloped viral particles leave the periplasmic space by fusion with the
outer nuclear envelope or the membrane of the endoplasmic reticulum
which is continuous with the nuclear envelope (Roizman and Knipe
2001; Mettenleiter 2002). By this procedure, capsids traverse the nuclear
envelope and reach the cytosol. The naked cytosolic capsids can recruit
tegument proteins and engage the host cytoskeleton to travel to the site
of secondary envelopment. Newly synthesised HSV1 particles tagged
with GFP-VP11/12 move along MTs over distances of up to 49 |im with
average velocities of 2 |im/s (Willard 2002).

In unpolarised epithelial cells, secondary envelopment most likely oc-
curs at the trans-Golgi network (TGN) or an endosome (Skepper et al.
2001; Mettenleiter 2002). In non-infected cultured cells, these organelles
are clustered around the MTOC in close proximity to the nucleus. Thus,
at least early during assembly, again dynein possibly assisted by dyn-
actin could transport the capsids to the desired location. However, later
during infection, the MTs are marginalised in bundles to the periphery

The Role of the Cytoskeleton During Viral Infection

A, Herpes Simple* Vim*

periplasm

nucleus

inner iuk"ltMr
mtmhrjik

cytoplasm
outer nuclear

membrane

B. Vaccinia Virus

viral factory

vi nipping compartment:

T(i\ orearlv encloNomc

IEV

ictin liliimriils

microtubuk-

200 ei in

Fig. 2A, B. Cytosolic transport during virus egress. A After nuclear HSV1 capsid as-
sembly and genome packaging, the capsid (dark green) acquires a primary envelope
by budding at the inner nuclear membrane. This primary envelope is lost by subse-
quent fusion with the outer nuclear membrane, and the capsid is released into the
cytosol. Most if not all tegument proteins (light green) are acquired in the cytosol or
during the second envelopment. In neurons, capsids with associated tegument and
membrane vesicles (grey) containing viral envelope proteins are transported along
microtubules (red) to the presynapse. Axonal transport is mediated by conventional
kinesin (dark blue). Presumably, secondary envelopment occurs in the nerve end-
ings. B The intracellular mature form (IMV) of vaccinia virus containing the core
(green) is assembled in viral factories located in the cytoplasm. The IMV is trans-
ported to the MTOC region along MTs by dynein (blue) and dynactin (light blue). At
the MTOC, the IMV is enwrapped by a membrane cisterna derived from the trans-
Golgi network or an early endosome, resulting in the formation of the intracellular
enveloped virus (IEV). The IEV requires MTs and conventional kinesin (dark blue)
for its transport to the plasma membrane. Shortly before or after fusion of the outer
IEV membrane with the plasma membrane, polymerisation of actin filaments
(orange) is induced. The cell-associated virus (CEV) is pushed on top of a long mi-
crovillus into the medium or a neighbouring cell

K. Dohner • B. Sodeik

of the cytoplasm, and there is no longer an obvious MTOC (Avitabile et
al. 1995; Elliott and O'Hare 1998, 1999; Kotsakis et al. 2001). The reor-
ganisation of the MT network most likely also changes the steady-state
distribution of the membrane organelles. The localisation of MT plus-
or minus-ends has not been determined late in infection, and therefore
it is difficult to predict which MT motors would then catalyse capsid
transport to the organelle of secondary envelopment.

In a typical neuronal axon, the MT plus-ends are uniformly oriented
towards the synapses and the minus-ends to the cell body (Goldstein
and Yang 2000). There are no reasons to believe that this organisation is
changed during a herpesvirus infection. Progeny virus spreads via the
axons, and the traditional model predicted that axonal transport of fully
assembled virions occurs inside vesicles. However almost 10 years ago
Cunningham and collaborators demonstrated that in human primary
sensory neurons HSV1 naked capsids were closely associated with ax-
onal MTs (Penfold et al. 1994). This study suggested that in neurons the
secondary envelopment might take place in the presynapse (Fig. 2A).

Immune labelling showed that the glycoproteins are transported in
vesicles, separately and with different kinetics from the capsids (Holland
et al. 1999; Miranda-Saksena et al. 2000; Ohara et al. 2000). In pseudora-
bies virus, the type II integral membrane protein US9 is required for tar-
geting of all tested viral membrane proteins to the axon, but not for ax-
onal transport of capsids and some tegument proteins (Tomishima and
Enquist 2001). Immunoelectron microscopy showed that progeny axonal
cytosolic capsids co-localise with conventional kinesin (Diefenbach et
al. 2002). As during virus entry, GFP- tagged pseudorabies virus is trans-
ported in both directions in axons late in infection. But during egress
the anterograde transport predominates. Average plus-end-directed ve-
locities are 2 um/s and minus-end directed velocities 1.3 |im/s (Smith et
al. 2001).

Thus, during axonal transport, progeny capsids recruit a plus-end
MT motor, most likely conventional kinesin. In epithelial cells a minus-
end-directed MT motor could ensure, at least early during assembly,
transport to the organelle of secondary envelopment.

5.3

Biochemical Analysis of Viral Capsid Transport

Several assays have been developed to characterise the host factors as
well as the viral receptors involved in MT transport. GFP-labelled cap-
sids were isolated after detergent extraction of purified virions (Bearer

The Role of the Cytoskeleton During Viral Infection 87

et al. 2000; Wolfs tein, Nagel, Dohner, Allan and Sodeik, in preparation).
Bearer et al. (2000) injected such capsids into the giant axon of the squid
Loligo paelei, a classic system to analyse fast MT-mediated transport.
HSV1 capsids labelled with co-packaged VP16-GFP that had been ex-
pressed from a complementing cell line moved in a continuous fashion
in retrograde direction with average velocities of 2.2 |im/s. This unidi-
rectional, non- saltatory movement is different to the motility of GFP-
tagged herpes particles after infection of vertebrate cells (Smith et al.
2001; Willard 2002; Dohner, Buttner, Wolfstein, Schmidt and Sodeik, in
preparation).

Wolfstein et al. (in preparation) reconstituted capsid motility in vitro
with Cy3-labeled MTs, VP26GFP-tagged capsids, an ATP-regenerating
system and a cytosolic protein fraction. In vitro transport was also salta-
tory, with long continuous runs of more than 10 jum in one direction,
and the capsids often changed MTs. In this assay capsid transport along
MTs depended on their tegument composition. It could be inhibited by
the addition of recombinant dynamitin, suggesting that the motility was
catalysed by dynein or heterotrimeric kinesin (Wolfstein, Nagel, Dohner,
Allan and Sodeik, in preparation). Such biochemical assays can now be
used to characterise the molecular interactions between viral and host
factors.

5.4

Interaction of Herpesvirus with Cytoskeletal Proteins

VP22, one of the major tegument proteins of HSV1, and GFP-VP22 co-
localise with MTs after transfection, whereas in infected cells VP22 does
not show a prominent enrichment on MTs (Elliott and O'Hare 1997,
1999; Kotsakis et al. 2001). The transient restructuring of the MT net-
work during virus infection coincides with the translocation of VP22
from the cytoplasm to the nucleus (Kotsakis et al. 2001). Transient ex-
pression of VP22 or infection with HSV1 leads to a stabilisation and hy-
peracetylation of MTs (Elliott and O'Hare 1998, 1999). A basic VP22
fragment contains the MT binding domain (Martin et al. 2002).

In addition, VP22 interacts with non-muscle myosin II, and the myo-
sin inhibitor BDM reduces virus yields (van Leeuwen et al. 2002). Be-
cause VP22 is released into the cytosol on HSV1 fusion with the plasma
membrane, this interaction could also facilitate the penetration of the
actin cortex by the incoming viral capsid.

The first herpesvirus protein reported to interact with the DIC sub-
unit of cytoplasmic dynein in GST pull down assays was the UL34 of

K. Dohner • B. Sodeik

HSV1, with a predicted type II integral membrane topology (Ye et al.
2000; Reynolds et al. 2002). UL34 of HSVand pseudorabies virus is tar-
geted to the inner nuclear membrane, where during budding it is incor-
porated into the primary envelope of periplasmic virions (Klupp et al.
2000; Reynolds et al. 2002). In contrast to the inner nuclear membrane,
cytoplasmic membranes and extracellular virions do not contain UL34;
thus it cannot be involved in viral cytoplasmic transport.

The heavy chain of conventional kinesin interacts with the HSV1
tegument protein US11. Moreover, US 11 and the major capsid protein
VP5 were reported to be transported with similar kinetics into the axons
of dissociated rat neurons (Diefenbach et al. 2002).

Retrovirus Entry and Budding

HIV, the most prominent representative of retroviruses, enters cells pri-
marily by membrane fusion at the plasma membrane (Greene and Peter-
lin 2002), although some strains can also infect cells via endocytic up-
take (Fackler and Peterlin 2000). During passage through the cytosol,
the viral RNA genome is reverse transcribed into DNA in a structure
named the reverse transcription complex (RTC) or pre-integration com-
plex (PIC). PIC is targeted to the nuclear pore complex and imported
into the nucleus, and the HIV genome is integrated into a chromosome
(Greene and Peterlin 2002; see the chapter by Byland and Marsh, this
volume, and Fig. IB).

Cytochalasin prevents the co-clustering of the HIV receptors CD4 and
CXCR4 on gpl20 binding and thus somehow inhibits efficient HIV fu-
sion with the plasma membrane (Iyengar et al. 1998). On cell fraction-
ation RTCs are localised in a cytoskeletal fraction, and reverse transcrip-
tion requires intact actin filaments (Bukrinskaya et al. 1998).

Intracellular trafficking of HIV can be visualised with a GFP-Vpr fu-
sion protein which is packaged into virions (McDonald et al. 2002). By
live cell imaging GFP-Vpr-labelled subviral particles co-localise with
MTs, move in curvilinear paths in the cytoplasm and accumulate around
the MTOC as early as 2 h after infection. HIV transport is completely
blocked in the presence of both nocodazole and latrunculin, but not by
either one alone. The incoming PIC of human foamy virus, another ret-
rovirus, also accumulates around the MTOC by a MT depending trans-
port (Saib et al. 1997).

The Role of the Cytoskeleton During Viral Infection 89

When dynein is inhibited by a microinjected antibody, the relative
transport of RTCs to the nucleus is reduced (McDonald et al. 2002). In-
terestingly, in this situation the RTCs accumulate in the cell periphery,
similar to what has been described for HSV capsids after overexpression
of dynamitin (Dohner et al. 2002). Moreover, yeast two-hybrid assays
showed an interaction of HIV integrase with the yeast homologue of dy-
nein LC8 (Richard de Soultrait et al. 2002).

Most likely, HIV subviral particles that are assembled in the cyto-
plasm also use active transport to reach the plasma membrane, where
retrovirus budding often takes place (Greene and Peterlin 2002). Both
MTs and the binding motive for the host factor hnRNP A2 present in ge-
nomic HIV RNA are involved in trafficking of RNA granules (Mouland
et al. 2001). The human T cell leukaemia virus, a retrovirus distantly re-
lated to HIV, requires a cell-cell junction and MTs for efficient transmis-
sion. T cell adhesion induces a rapid reorganisation of the MT network
towards the junction that becomes a kind of virological synapse for bud-
ding (Igakura et al. 2003).

Filamentous actin and myosin seem to play important roles during
HIV budding at the plasma membrane (Sasaki et al. 1995). The interac-
tion of the HIV negative effector Nef with Vav, the host guanine nucleo-
tide exchange factor of Cdc42 and Rac — two small GTPases regulating
the actin cytoskeleton — could modify the actin cortex before virus bud-
ding (Fackler et al. 1999). Nef is also packaged into virions (10-100 mo-
lecules/particle) and has an important role in viral entry early after virus
fusion but before the completion of reverse transcription (Schaeffer et
al. 2001; Forshey and Aiken 2003). It is tempting to speculate that Nef,
possibly in concert with other proteins of the RTC, could again remodel
the actin cortex to facilitate access to the MT plus-ends for further
transport.

The kinesin motor KIF4A is abundantly expressed in neurons and
transports vesicles containing the cell adhesion protein LI (Peretti et al.
2000). Yeast two-hybrid assays and chromatography showed that Gag
polyproteins and matrix protein of murine leukaemia virus, HIV and
SIV bind to KIF4 (Kim et al. 1998; Tang et al. 1999). Such an interaction
could mediate a plus-end-directed transport of the RTC during entry, as
well as the transport of RNA granules to the plasma membrane for as-
sembly.

K. Dohner • B. Sodeik

Adenovirus Entry and Microtubules

The cell entry of adenovirus is characterised by the stepwise disassem-
bly of incoming virions (Greber et al. 1993; Fig. 1C). Adenoviruses are
internalised by receptor-mediated endocytosis and induce the lysis of
endosomal membranes to gain access to the cytosol (Shenk 2001). Ad2
and Ad5 virions of the subgroup C attach to MTs in vitro and in vivo
(Luftig and Weihing 1975; Miles et al. 1980). If cells have been treated
with vincristine to induce MT paracrystals, the virions display projec-
tions emanating from the vertices that are shorter and thicker than the
fibre, and thus might represent bound host factors (Dales and Chardon-
net 1973). The capsids use dynein for transport along MTs towards the
MTOC because depolymerising the MT network, overexpressing dyna-
mitin and microinjection of function-blocking dynein antibodies all in-
hibit Ad2 and Ad5 infection (Suomalainen et al. 1999; Leopold et al.
2000; Mabit et al. 2002).

As with herpesvirus, it is unclear whether and how viral capsids
which have reached the MTOC are forwarded to the nuclear pores. Dy-
namic MTs are not required for nuclear targeting of incoming Ad parti-
cles. In fact, suppressing MT dynamics enhances the nuclear targeting
efficiency of Ad2 (Giannakakou et al. 2002; Mabit et al. 2002). At the nu-
clear pores the viral DNA is injected into the nucleoplasm (Greber et al.
1993; Trotman et al. 2001), where viral replication, transcription and
capsid assembly take place.

Analysing Ad2 particles that were visualised by the covalent attach-
ment of fluorescent dyes, Suomalainen et al. (1999) described two types
of motilities. Several transport events last for several seconds with an av-
erage velocity of 0.3-0.5 |im/s, depending on the cell type, and peak ve-
locities of up to 2.6 |im/s which are directed exclusively towards the
MTOC. Other particles rapidly alter direction towards or away from the
nucleus with speeds of up to 0.5 |im/s. As with many other viruses, the
incoming Ad2 particles induce a complex signalling cascade, which in
this case activates protein kinase A and p38/MAPK pathways to boost
minus-end-directed MT capsid transport (Suomalainen et al. 2001). In
contrast to Ad2, Leopold et al. (1998, 2000) showed that labelled Ad5
particles rarely switch directions but also move along curvilinear tracks
with an average speed of 2.2 |im/s. In the absence of a MT network, both
groups detected only short-range random movements (Suomalainen et
al. 1999; Leopold et al. 2000).

The Role of the Cytoskeleton During Viral Infection 91

Glotzer et al. (2001) labelled Ad5 particles by co-packaging a GFP-la-
belled protein that bound to specific sequences engineered into the viral
genome. Such particles moved both towards and away from the nucleus
and, surprisingly, with comparable peak velocities of 1 |im/s and average
velocities of 0.32 |im/s or 0.26 |im/s, irrespective of the presence or ab-
sence of MTs. Moreover, viral gene expression is not reduced if MTs have
been depolymerised before infection (Glotzer et al. 2001). To explain
these conflicting results on cytosolic adenovirus capsid transport, the
parallel analysis of differently labelled particles with an identical experi-
mental set-up and time resolution will be necessary (for a discussion,
see Mabit et al. 2002).

The adenoviral protein Ad E3-14.7K, an inhibitor of TNFa-induced
cell death, interacts with a small GTPase called FIP1, which in turn inter-
acts with TCTEL1, a human homologue of the DLC Tctex-1 (Lukashok et
al. 2000). Because Ad E3-14.7K is not a structural protein, it cannot have
a role in cytosolic transport of Ad particles. However, many cellular pro-
teins require MT-mediated transport to reach their final destination
(Giannakakou et al. 2000), and the interaction of Ad E3-14.7K with a
FIP1-TCTEL1 complex could aid in its cytosolic trafficking.

Altogether these studies suggest that adenovirus capsids possess a vi-
ral receptor either for dynein or for dynactin, which allows them to use
MTs for efficient transport to the host nucleus after exit from the endo-
some. The disruption of the intermediate filament network facilitates vi-
ral egress (Chen et al. 1993), and progeny adenoviruses are released by
cell lysis.

Parvovirus Entry and Microtubules

Parvoviruses enter cells by receptor-mediated endocytosis and seem to
cross endosomal membranes at different stages of the endocytic path-
way. Canine parvovirus (CPV) is detected in clathrin-coated vesicles, re-
cycling endosomes, late endosomes and finally lysosomes, and CPV-con-
taining vesicles are often associated with MTs. The transport from early
to late endosomes as well as that of CPV-containing vesicles to the peri-
nuclear area requires intact MTs and cytoplasmic dynein (Aniento et al.
1993; Suikkanen et al. 2002). Injected anti-capsid antibodies block CPV
infection, suggesting that capsids enter the cytosol during infection.
Moreover, nocodazole and a microinjected anti-dynein antibody block
the nuclear accumulation of the injected capsids, suggesting that cyto-

K. Dohner • B. Sodeik

solic CPVare transported by dynein along MTs to the nucleus (Suikkanen
et al. 2003).

Adeno-associated virus (AAV) particles also move in a nocodazole-
and cytochalasin B-sensitive manner to the nucleus. However, it remains
to be determined whether virions are transported inside vesicles or
freely in the cytosol (Sanlioglu et al. 2000). Real-time imaging of viral
particles labelled with a single fluorescent molecule shows diffusion of
free virions, diffusion of virus within endosomes and directed MT-
dependent motion with velocities of 1.8-3.7 um/s (Seisenberger et al.
2001).

Poxviruses — Multiple Cargos for Microtubules and Actin Tails
9.1

Cytosolic Vaccinia Virus Cores

The most complicated virus in terms of entry and assembly is vaccinia
virus (VV), a poxvirus. One of its unique features is the formation of
two infectious viral particles. Assembly commences in viral factories
leading to the formation of the intracellular mature virus (IMV; Sodeik
et al. 1993), a large enveloped brick-shaped particle. Relative to the IMV,
the extracellular enveloped virus (EEV) has one additional membrane,
the so-called envelope (Smith et al. 2002; Sodeik and Krijnse-Locker
2002).

Because both forms lead to infection, they must differ in how the
DNA genome is uncoated and released into the cytosol where viral repli-
cation takes place. There are conflicting reports on whether both EEV
and IMV, or only IMV, require actin filaments for entry and whether
both or only IMV enter directly at the plasma membrane rather than by
endocytosis (Payne and Norrby 1978; Doms et al. 1990; Vanderplasschen
et al. 1998). In a recent study, the cells responded only to IMV but not
EEV with the formation of long cell surface protrusions which were in-
duced by a signalling cascade involving the actin-binding protein ezrin
and the small GTPase Rac (Krijnse Locker et al. 2000). Accordingly, the
IMV but not the EEV requires actin dynamics and filopodia for entry at
the plasma membrane.

Although VV entry is still poorly understood, at the end of this pro-
cess cores lacking any membrane are delivered into the cytosol. Early
VV mRNAs are first transcribed inside these cores and then accumulate

The Role of the Cytoskeleton During Viral Infection 93

in large granular structures which recruit ribosomes for translation and
are some distance away from the cores (Mallardo et al. 2001). Both the
cores and the early mRNAs are associated with MTs, and nocodazole re-
duces mRNA synthesis (Mallardo et al. 2001). The in vitro binding of
isolated cores to MTs requires the DNA- and RNA-binding protein L4R
and the core protein A10L, which both co-localise with stabilised MTs in
infected cells (Ploubidou et al. 2000; Mallardo et al. 2001).

Next, the incoming genome is uncoated, released from the core and
amplified in specialised replication sites that are surrounded by mem-
branes of the endoplasmic reticulum (Tolonen et al. 2001). After synthe-
sis of the structural proteins from the late mRNAs, IMVs are formed
within cytoplasmic viral factories from non-infectious precursors called
crescents and immature virions (Fig. 2B).

9.2

Particle Transport During Virus Egress

Overexpression of dynamitin, or the deletion of the IMV protein A27L,
inhibits the minus-end-directed MT transport of IMV particles from the
viral factories to the MTOC (Ploubidou et al. 2000; Sanderson et al.
2000; Fig. 2B). At the MTOC, the IMV is wrapped in a membrane cister-
na derived from the -TGN or an early endosome and intracellular en-
veloped virus (IEV) with two additional membranes relative to the IMV
is formed (Smith et al. 2002). If cells are infected in the presence of no-
codazole, no IEVs are made and there is a threefold reduction in the
amount of IMVs synthesized (Ploubidou et al. 2000).

IEVs move to the plasma membrane in a saltatory and directional
manner with an average speed of 1-2 jimls along MTs and even switch
from one MT to another (Geada et al. 2001; Hollinshead et al. 2001;
Rietdorf et al. 2001; Ward and Moss 2001). IEVs co-localise with ki-
nesin-1, and the overexpression of the cargo-binding domain of KLC in-
hibits their transport (Rietdorf et al. 2001). Besides kinesin, the integral
membrane protein A36R and the peripheral membrane protein F12L are
necessary for IEV transport to the plasma membrane (Rietdorf et al.
2001; van Eijl et al. 2002).

Shortly before or after the fusion of the outer membrane of IEV with
the plasma membrane, the polymerisation of actin filaments is induced
(Hollinshead et al. 2001). The cell-associated virus (CEV) that remains
attached to the plasma membrane for quite some time is pushed on top
of a long microvillus into the medium or a neighbouring cell at a speed
of 2.8 jum/s (Cudmore et al. 1995). This process facilitates VV spread in

K. Dohner • B. Sodeik

a cell monolayer and in the tissue. The IEV protein A36R, which has an
unusual long cytosolic tail and no obvious mammalian homologues, is
the vaccinia actin tail nucleator. Tyr phosphorylation of A36R by a
member of the Src-kinase family leads to the recruitment of the adaptor
proteins Nek and Grb2, the WASP-interacting protein (WIP) as well as
N-WASP (Wiskott-Aldrich syndrome protein). WASP activates the Arp2/
3 complex and thus motility based on actin polymerisation (Frisch-
knecht et al. 1999; Moreau et al. 2000; Scaplehorn et al. 2002). Another
interesting rearrangement of the host actin cytoskeleton is the induction
of cell migration that depends on early VV gene expression and the for-
mation of cellular projections up to 160 jum in length that requires late
VV gene expression (Sanderson et al. 1998).

Considering how many different types of large VV particles have to
be shuffled around during entry and assembly, it is probably not sur-
prising that there is considerable cross-talk between VV proteins and
both cytosolic transport systems, MTs and microfilaments.

Actin Remodelling During Baculovirus Infection

Baculoviruses such as Autographa californica M nuclear polyhedrosis vi-
rus (AcMNPV) catalyse several distinct rearrangements of the actin cy-
toskeleton during different stages of the viral infection.

After virus uptake by endocytosis and acid-induced endosomal es-
cape, the incoming cytosolic capsids induce prominent transient actin
cables which often have a single capsid at one end and seem to be in-
volved in capsid transport to the nucleus (Charlton and Volkman 1993;
van Loo et al. 2001). Isolated AcMNPV capsids can nucleate actin poly-
merisation in vitro, and competition data with cytochalasin suggest
that these capsids bind to the minus-end of the filaments (Lanier and
Volkman 1998). Two AcMNPV proteins, p39 and p78/83, can bind to
actin directly (Lanier and Volkman 1998). Moreover, p78/83 contains
regions of homology to WASP which bind monomeric actin and the
Arp2/3 complex which nucleates assembly of new actin filaments (Mach-
esky et al. 2001). This suggests that p78/83 could function as a viral
WASP homolog in inducing actin filament assembly.

Interestingly, in contrast to other viruses, depolymerising the MTs
with nocodazole speeds up the onset of baculovirus gene expression, at
least in transduction experiments using mammalian cells, whereas infec-
tion in the presence of cytochalasin reduces the infection efficiency (van

The Role of the Cytoskeleton During Viral Infection 95

Loo et al. 2001). The myosin inhibitor BDM inhibits capsid transport to
the nucleus, suggesting that a myosin could also be involved in capsid
transport (Lanier and Volkman 1998).

The expression of the early viral protein Arif-1 (actin rearrangement-
inducing factor 1) is responsible for second modification of the actin cy-
toskeleton, the appearance of actin aggregates localised at the plasma
membrane (Roncarati and Knebel-Morsdorf 1997). The amino acid se-
quence of Arif-1 predicts a N-terminal signal peptide and three trans-
membrane regions of about 20 amino acids each, and, moreover, Arif-1
seems to be phosphorylated at tyrosine residues (Dreschers et al. 2001).
Arif- 1 is not required for infection of cultured cells but might play a role
during pathogenesis in insects.

The later phases of baculovirus infection are characterized by the ap-
pearance of F-actin inside the nucleus in a ring close to the nuclear
membrane which is essential for nucleocapsid morphogenesis (Ohkawa
and Volkman 1999). Six gene products of AcMNPV are required for the
recruitment of G-actin to the nucleus (Ohkawa et al. 2002).

Perspectives

There is probably not a single virus for which the cytoskeleton does not
play important roles during entry, replication, assembly or egress. With
the emerging technologies in digital time-lapse microscopy, the further
characterisation of viral transport will improve our understanding of
the underlying host mechanisms.

Deciphering the molecular interactions between viruses and the cyto-
skeleton could provide important new targets for the development of an-
tiviral therapy. Although the cytoskeletal filaments and associated mo-
tors play major roles in the cellular metabolism, viruses might interact
with these structures via unique sites different from the cellular interac-
tion partners. Other potential targets might be specific host proteins
which viruses use to hook onto the cytoplasmic transport machinery.

In our quest to develop efficient vectors for therapeutic gene expres-
sion, it has become apparent that the transport of naked DNA to the nu-
cleus, where transcription usually takes place, is a major barrier for the
transfection of non-dividing cells, and hence for the application of hu-
man non-viral gene therapy vectors in vivo. However, several viruses
have found effective solutions to this problem, namely successful negoti-
ation with the host cytoskeleton to ensure efficient cytoplasmic trans-

K. Dohner • B. Sodeik

portation to the nuclear pores of the nuclear envelope. The future design
of viral vectors will include these specific nuclear targeting mechanisms
while deleting any factors responsible for cell toxicity and the induction
of the immune response.

Acknowledgements We thank Rudi Bauerfeind (Cell Biology, MHH) and Elena
Korenbaum (Biophysical Chemistry, MHH) for many helpful suggestions on the
manuscript and Oliver Fackler (Virology, University of Heidelberg) for insightful
discussions on HIV cell entry.

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© Springer- Verlag 2004

Nuclear Import in Viral Infections

U. R Greber 1 (^) • M. Fornerod 2

1 Zoologisches Institut der Universitat Zurich, Winterthurerstrasse 190,
8057 Zurich, Switzerland

ufgreber@zool.unizh.ch

2 The Netherlands Cancer Institute H4, Plesmanlaan 121, 1066 CX, Amsterdam,
The Netherlands

1 Introduction

110

2 Nuclear Pore Complexes, Guardians of the Nucleus Ill

3 Soluble Import Receptors Control Nuclear Access 113

4 Models of Nuclear Pore Complex Translocation 114

5 Ran-Independent Transport 116

6 Nuclear Import of Viral Genomes 117

6.1 Parvoviruses 118

6.2 Hepadnaviruses, DNA Retroviruses 119

6.3 Retrotransposons 120

6.4 Lentiviruses 121

6.5 Adenoviruses 123

6.6 Herpesviruses 124

6.7 Papovaviruses 125

6.8 Negative-Sense RNA Viruses 127

7 Perspectives 128

References

129

Abstract The separation of transcription in the nucleus and translation in the cyto-
plasm requires nucleo-cytoplasmic exchange of proteins and RNAs. Viruses have
evolved strategies to capitalize on the nucleo-cytoplasmic trafficking machinery of the
cell. Here, we first discuss the principal mechanisms of receptor-mediated nuclear im-
port of proteinaceous cargo through the nuclear pore complex, the gate keeper of the
cell nucleus. We then focus on viral strategies leading to nuclear import of genomes
and subgenomic particles. Nucleo-cytoplasmic transport is directly important for
those viruses that are replicating in the nucleus, such as DNA tumor viruses and RNA
viruses, including parvoviruses, the DNA retroviruses hepadnaviruses, RNA-retro-
transposons and retroviruses, adenoviruses, herpesviruses, papovaviruses, and partic-
ular negative-sense RNA viruses, such as the orthomyxovirus influenza virus. The vi-
ral strategies of nuclear import turn out to be surprisingly diverse. Their investigation
continues to give insight into how nucleic acids pass in and out of the nucleus.

110

U.F. Greber • M. Fornerod

Introduction

Viruses interfere with all aspects of cell physiology. This can lead to lytic
infections releasing large amounts of progeny particles from the infected
cells, persistent infections producing moderate amounts of progeny, or
latent infections without apparent particle production. All of these pos-
sible outcomes can ensure viral survival and spread. How viruses inter-
act with their hosts has largely been selected for by the pressure of the
immune system and the underlying cell biological machineries. Normal-
ly, host cells are protected from infections by multiple barriers during
entry, replication, and viral release. Major hurdles for the incoming
viruses are the extracellular matrix and the accessibility of receptors, the
plasma membrane, the cytoplasm, and the nuclear membrane. An effec-
tive strategy to overcome the extracellular matrix is to tightly bind to a
receptor in the plasma membrane (Wimmer 1994). Interestingly, some
viruses have chosen to use a receptor that is cryptic, that is, not readily
accessible from the apical lumen of polarized cells where incoming res-
piratory or enteric viruses initiate their infections (Spear 2002; Stehle
and Dermody 2003). Examples of viral receptors localized in cell junc-
tions include the coxsackievirus B adenovirus (Ad) receptor (CAR) serv-
ing as a high-affinity binding site for most Ad serotypes, the nectin-1 re-
ceptor of alpha herpesviruses, and the junctional adhesion molecule
(JAM) of reoviruses. For those viruses the extracellular matrix is a par-
ticular challenge. One way to overcome it is to use the opportunity of
entry sites where the integrity of the epithelial barrier is disrupted or
otherwise nonpolarized cells are facing the lumen. Although it is not en-
tirely clear why this viral strategy has been so successful, it was recently
suggested for Ads that newly synthesized fiber proteins can disrupt cell-
cell contacts, loosen up the epithelial integrity, and aid viral release from
infected basal cells into the apical lumen (Walters et al. 2002). This may
be potentiated by the ability of an additional viral protein, penton base,
to abduce fiber from the infected to the surrounding noninfected cells
(Trotman et al. 2003). Thus CAR may serve two different viral functions,
as an exit and an entry receptor. Once an incoming virus has bound to a
cell receptor, it may catch a ride in endocytic vesicles shuttling from the
plasma membrane to the interior of the cell (see the chapter by
Sieczkarski and Whittaker, this volume). Other viruses have evolved
strategies to penetrate the plasma membrane or endosomes after receiv-
ing appropriate signals from the cell (see the chapter Earp et al., this vol-
ume). Alone, the delivery of the viral nucleic acid in the cytosol is in

Nuclear Import in Viral Infections

111

most cases not sufficient for replicating the genome or maintaining it in
a stable state within the host. Regulated cytoplasmic trafficking and nu-
clear deposition of an encapsidated genome is in many cases important
to ensure efficient access to the replication site (see the chapter by Pi-
guet, this volume). Viral replication and particle production can lead to
vertical transmissions, for example, in the case of papillomaviruses
spreading from the infected epithelium of the mother to the newborn.
Alternatively, horizontal transmissions can disseminate the viral parti-
cles throughout a population. This requires efficient entry, genome pro-
duction, and packaging processes as well as effective viral egress and
suppression of host antiviral defense reactions.

Clearly, all of these steps have evolved through an intimate relation-
ship of the virus with its host, and in many instances they depend on
the cell nucleus (for recent reviews on nuclear import of incoming virus-
es, see Greber and Fassati 2003; Whittaker 2003). In this review, we sum-
marize the cornerstones of nuclear import of incoming viral genomes
and highlight some of the better-studied postentry strategies of viruses
co-opting the cellular mechanisms of nucleo-cytoplasmic exchange.

Nuclear Pore Complexes, Guardians of the Nucleus

The nuclear envelope is the limiting feature of the nucleus harboring the
large majority of the genomic information of the cell. It contains ran-
domly positioned nuclear pore complexes (NPCs) controlling the ex-
change of cytosolic and nuclear macromolecules (Nakielny and Dreyfuss
1999). NPCs are large protein structures of approximately 125 MDa,
about 25 times the mass of a ribosome (Reichelt et al. 1990). They have
an eightfold symmetry if viewed in projection from the cytoplasmic or
the nuclear side (reviewed in Stoffler et al. 1999; Wente 2000). Ultra-
structural analyses using transmission electron microscopy (TEM),
scanning EM, and atomic force microscopy have shown that the verte-
brate NPC is a ~65-nm-deep channel, decorated on each side with eight
filaments attached to a coaxial ring that project away from the nuclear
envelope (reviewed in Allen et al. 2000; Fahrenkrog et al. 2001). The na-
ture of a central plug is controversial. It can either be an integral part of
the NPC or cargo in transit (for a recent discussion, see Suntharalingam
and Wente 2003). The ~50-100-nm filaments of the nuclear face join to-
gether at their distal ends to form a so-called nuclear basket or fish trap.

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U.F. Greber • M. Fornerod

The vertebrate NPC is built of 30-40 different proteins, termed nucle-
oporins (Cronshaw et al. 2002; Fahrenkrog et al. 2001; Rout et al. 2000;
Ryan and Wente 2000). Nucleoporins are proteins whose steady-state
distribution is predominantly in the NPC and that are somehow in-
volved in building the NPC or serving the NPCs transport function
(Vasu and Forbes 2001). A motif characteristic for nucleoporins is the
FG-repeat consisting of a large number of phenylalanine-glycine dipep-
tides, spaced by 4-20 intervening amino acids. These FG-repeat regions
have been shown to interact with transport receptors of the karyo-
pherin/importin type, as well as with the unrelated mRNA export recep-
tor NFX1/TAP complexed to pl5 (see below). In vertebrates, FG-repeat
nucleoporins have been localized to different positions of the NPC struc-
ture, such as the nuclear face (Nupl53), the cytoplasmic face (CAN/
Nup214), the cytoplasmic filaments (RanBP2/Nup358), and the central
channel (p62, Poml21). The exact localization of most other vertebrate
nucleoporins remains to be determined. The vertebrate NPCs are rela-
tively static in the plane of the nuclear membrane (Daigle et al. 2001),
presumably because they are bound to the lamina network at the inner
nuclear membrane. Nonetheless, evidence is emerging to indicate that
individual nucleoporins can be dynamic and shuttle between intranucle-
ar or cytoplasmic pools. For example, the NPCs of the single cell algae
Chlamydomonas reinhardtii were observed to move to the posterior pole
on deflagellation of the cell (Colon-Ramos et al. 2003). Increased levels
of /^-tubulin accumulate in the cytoplasm proximal to the rearranged
NPCs near the site where the new flagella will be built, suggesting a link
between cytoplasmic transcript localization and nuclear envelope archi-
tecture, in particular NPC localization. Interestingly, non-NPC proteins
are found to transiently associate with NPCs, for example, proteins in-
volved in cell cycle control and in protein modification with the small
ubiquitin modifier (SUMO) peptide that mediates protein-protein inter-
actions. In vertebrate cells, RanBP2/Nup358 has an E3 ligase activity
that transfers SUMO to acceptor proteins (Pichler et al. 2002). The
Ran:GTPase activating protein (RanGAPl) is sumoylated and docks at
the NPC. Additionally, it was found that the SUMO conjugating enzyme
Ubc9 can associate with RanGAPl and RanBP2/Nup358 and the SUMO
protease SENP2 localizes to the NPC by interacting with Nupl53 (Hang
and Dasso 2002; Zhang et al. 2002). It is possible that the SUMO machin-
ery at the NPC modifies particular cargoes in transit between the nucle-
us and cytoplasm (Greber and Carafoli 2002; Rodriguez et al. 2001). Yet
another level of NPC regulation may be imposed by phosphorylation. It
was found that in vitro reconstituted nuclear protein import is inhibited

Nuclear Import in Viral Infections

113

by the phosphatase inhibitor okadaic acid and partially reversed by ki-
nase inhibitors without affecting the association of import substrates
with the NPC, suggesting that phosphorylation of NPC components in-
hibits nuclear import (Kehlenbach and Gerace 2000). These results, al-
though preliminary, suggest that activation of specific cellular signaling
pathways can influence the number and perhaps the size of the mole-
cules that can cross the NPC. It emerges that the NPC integrates modifi-
er and transport functions of high capacity and high selectivity (Gorlich
and Kutay 1999). Thus the NPC seems to be an ideal target of viruses
aiming to subvert cellular control functions.

Soluble Import Receptors Control Nuclear Access

Efficient macromolecular trafficking across the NPC is both signal- and
receptor dependent. Nuclear localization (NLS) or nuclear export (NES)
signals on cargo proteins are recognized by transport receptors of the
karyopherin/importin j3 family, of which there are currently more than
20 members in vertebrates (reviewed by Fornerod and Ohno 2002; Gor-
lich and Kutay 1999; Strom and Weis 2001). Some of them are expressed
in a tissue- and developmentally restricted manner and can also serve
functions other than those required for nuclear transport (see, e.g.,
Geles et al. 2002). The prevalent function of importin and exportin su-
perfamily proteins in interphase cells is to mediate nucleo-cytoplasmic
transport of proteins, either free or bound to nucleic acids. Importins
are characterized by their ability to bind cargo and the small GTPase
Ran loaded with GTP (Ran:GTP), and they interact with the NPC. Al-
though most importins and exportins are rather specialized, recognizing
a restricted set of cargoes, the import receptor importin (5 transports a
wide variety of cargo, including viral proteins. Importin j5 can cooperate
with the adaptor importin a and with other importin family members,
and it serves to import proteins bearing classic SV40-type basic NLS
and also uridine-rich small nuclear ribonucleoproteins (snRNPs) (re-
viewed by Kuersten et al. 2001). Import complexes are allowed to form
in the cytoplasm, where the GTPase activating enzyme Ran GAP 1 re-
stricts the levels of Ran:GTP and the nuclear transport factor (NTF) 2
transports Ran:GDP into the nucleus. Inside the nucleus, Ran:GTP is re-
plenished by the chromatin-bound factor RCC1, exchanging GTP for
GDP in the nucleotide binding pocket of Ran. Ran:GTP dissociates the

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importin-cargo complex. The cargo is distributed to subnuclear sites,
and the receptors are recycled to the cytoplasm.

Models of Nuclear Pore Complex Translocation

NPCs mediate two types of translocation, passive diffusion of small sub-
stances and receptor-dependent translocations of signal-bearing car-
goes. The size exclusions of both transport modes are kinetically deter-
mined. That is, within a time frame of a few hours the largest cargoes
seen to diffuse through the NPC have been about 9 nm in diameter (re-
viewed in Gerace and Burke 1988). The largest cargoes of facilitated
transport are 39-nm gold particles coated with the nucleophilic protein
nucleoplasmin and the transport factors importin a and j3 (Pante and
Kann 2002). Furthermore, the rates of substance flux through an indi-
vidual NPC are considerable, namely, in the order of 1,000 cargo mole-
cules per second (Ribbeck and Gorlich 2001; Smith et al. 2002).

One model of translocation proposes that increasing affinities be-
tween nucleoporins and transport receptors along the distance of the
NPC determine the direction of translocation (Bednenko et al. 2003).
This model is supported by the strong interaction of the import receptor
importin j3 to the FG repeat region of Nupl53 located at the nuclear bas-
kets (Shah et al. 1998) and the strong interaction of the export receptor
CRMl/exportinl to the FG repeat region of the nucleoporin CAN/
Nup214 located at the cytoplasmic filaments (Askjaer et al. 1999; Kehlen-
bach et al. 1999). Nupl53 and CAN/Nup214 could therefore represent
the terminal and highest- affinity binding sites of an import and export
reaction, respectively (Ben-Efraim and Gerace 2001). Several lines of evi-
dence argue against the affinity gradient being the sole force of direc-
tional transport within the NPC. First, directionality can be reversed by
the reversal of the Ran:GTP gradient across the nuclear envelope, indi-
cating that the relative binding strengths of nucleoporins with receptors
are not necessary for directionality of transport (Nachury and Weis
1999). This argues that the Ran:GTP gradient and the associated stabili-
ties of cargo-transport factors are sufficient to achieve vectorial translo-
cation of cargo. Second, the translocation would be driven by binding to
the highest-affinity site and release by binding of nuclear Ran:GTP (im-
port) or Ran:GTP hydrolysis (export). However, nuclear import can oc-
cur in the absence of GTP hydrolysis on Ran (Schwoebel et al. 1998),
and single rounds of import or export can occur in the absence of Ran

Nuclear Import in Viral Infections

115

(Englmeier et al. 1999; Huber et al. 2002; Ribbeck et al. 1999). In addi-
tion, the export and reimport rates of a complex consisting of the export
factor CRM1, NES- containing cargo, and Ran:GTP in Xenopus oocytes
are similar (Becskei and Mattaj 2003), further suggesting that the affinity
gradient model may only partly account for the observed transport di-
rectionalities.

On the basis of kinetic experiments a second model has proposed that
the FG repeats of nucleoporins form a hydrophobic meshwork within
the central NPC channel that is restrictive for most proteins because of
their hydrophilic surfaces (Ribbeck and Gorlich 2001). The model pre-
dicts that transport receptors immerse into this meshwork by virtue of
their rather hydrophobic surface and diffuse through the NPC channel.
Supporting evidence for this model shows that import receptors bind to
hydrophobic interaction resins and the permeability barrier of the NPC
can be compromised by the addition of hexandiole, which is thought to
weaken or break up the meshwork (Ribbeck and Gorlich 2002). This hy-
pothesis can potentially explain the nature of the diffusion barrier
through the central channel of the NPC, even though it cannot explain
the specific role of some nucleoporins for specific transport pathways,
for example, Nup82 for mRNA export (Grandi et al. 1995; Hurwitz and
Blobel 1995) and Nupl53 for NLS-protein import (Walther et al. 2001).

In a third model, called the "virtual gate" (Rout et al. 2000), filamen-
tous nucleoporins have been suggested to play a major role in protein
translocation. In this scenario, the random Brownian movement of the
cytoplasmic filaments would clear away noncargo proteins from the en-
trance of the NPC channel. Importin-cargo complexes on the other hand
would be concentrated by binding to the cytoplasmic filaments and sub-
sequently diffuse through the central translocation channel into the nu-
cleus, where they would be disassembled by Ran:GTP (Rout et al. 2000).
This model is attractive, but it still awaits experimental support. Nucleo-
porin FG repeats are likely to be in an unstructured conformation and
therefore indeed could be considered as microfilaments (Denning et al.
2003). Interestingly, however, the removal of the cytoplasmic filaments
of the NPC does not reduce NLS import or increase the passive perme-
ability of the NPC in vitro (Walther et al. 2002). Together, all three mod-
els agree that the interactions of transport factor and nucleoporin FG re-
gions are key for translocation through the NPC, but none of the models
alone can yet explain all the observed features of nucleo-cytoplasmic
transport. Of particular interest here is the observation that entry of
large cargo into the NPC alone is apparently not sufficient for transloca-
tion (Lyman et al. 2002) and that the translocation efficiency correlates

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U.F. Greber • M. Fornerod

with the number of bound transport factors (Ribbeck and Gorlich
2002). This is particularly interesting in the light of a recent biophysical
analysis of the NPC by atomic force microscopy suggesting that trans-
port receptors with a relatively weak affinity to the FG repeats of the nu-
cleoporins lead to a dilation of the pore whereas high-affinity receptors
seem to promote an expansion of the pore in the vertical direction (Jaggi
et al. 2003). This all suggests that diffusion alone, particularly of large
cargo, may not be sufficient for NPC translocation.

Ran-lndependent Transport

Importins and exportins do not require the Ran system to enter or exit
the nucleus, presumably because of their interactions with nucleoporin
FG repeats (Kose et al. 1997, 1999; Nakielny and Dreyfuss 1998; Ribbeck
et al. 1999). Importins and exportins consist of approximately 20 repeats
of the so-called HEAT or Armadillo type (Andrade et al. 2001). Interest-
ingly, /?-catenin and importin a, which have a similar overall structure,
are also able to move in and out of the nucleus independent of Ran
(Henderson and Fagotto 2002; Miyamoto et al. 2002). /?-Catenin is a
component of cell-cell adhesion junctions binding directly to cadherins
and linking to the actin cytoskeleton via a-catenin. The biological activ-
ity of /?-catenin is controlled by the wnt signaling pathway and in partic-
ular by the adenomatous polyposis coli protein, a predominantly cyto-
plasmic factor that binds and thereby facilitates /?-catenin degradation.
Failure of /?-catenin degradation and concomitant nuclear accumulation
of /?-catenin are associated with colon cancer. /?-Catenin contains no rec-
ognizable NLS, and it was found to be imported into nuclei of permeabi-
lized cells in the absence of transport factors and Ran (Fagotto et al.
1998; Yokoya et al. 1999). Whether y8-catenin has any shuttling function
like the importin factors is not known.

Another example of importin-independent transport is the MAP ki-
nase ERK. ERKs play a key role in transducing extracellular signals into
physiological cell responses during proliferation, differentiation, and
early embryonic development. There are at least two pathways known
for ERK nuclear import, Ran-dependent transport of dimeric phosphor-
ylated ERK (Khokhlatchev et al. 1998) and passive diffusion of mono-
meric ERK (Adachi et al. 1999). In digitonin-permeabilized cells, GFP-
fused ERK accumulates in the nucleus independently of phosphorylation
and in the absence of soluble factors and ATP (Matsubayashi et al.

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117

2001). This is inhibited by wheat germ agglutinin or by an excess of im-
portin j5 at reduced temperature, suggesting that nuclear accumulation
of GFP-ERK requires NPCs. ERKwas found to bind the carboxy- terminal
FG repeats of CAN/Nup214 in vitro, suggesting that it could interact di-
rectly with the NPC. This implies that the importin-independent translo-
cation depends on specific interactions of ERK and the NPC, distinct
from the interactions during importin-dependent translocation of di-
meric ERK (Shibayama et al. 2002). It will be interesting to see how
these distinct nuclear import pathways of ERK connect to particular sig-
nal transduction pathways.

Interestingly, nuclear localization of certain large substrates, namely
the spliceosomal U snRNP particles has been reported to be Ran inde-
pendent (Huber et al. 2002; Rollenhagen et al. 2003). It is mediated by
the adaptor protein snurportin, which binds to the m3G-cap structure
at the 5' end of the U RNAs and, in addition, has an importin j3 binding
domain (IBB). Importin j3 and snurportin were sufficient for a single
round of import of Ul snRNPs in digitonin-permeabilized mammalian
cells and did not require Ran and trinucleotides, such as ATP or GTP.
When the IBB domain of snurportin was exchanged with the IBB do-
main of importin a, nuclear import of Ul snRNPs was dependent on
Ran. This suggests that different IBB domains influence interactions of
importin (5 with nucleoporins of the NPC and determine whether Ran is
required to dissociate these interactions. Interestingly, the size of the
cargo did not seem to be a major factor impeding translocation in these
experiments, because the U5 snRNP with a molecular mass larger than
one million daltons was also imported in a Ran- and energy-indepen-
dent manner when native snurportin was present.

Nuclear Import of Viral Genomes

Viruses that replicate in the nucleus of nondividing cells with an intact
nuclear membrane transfer their genomes through the NPC into the nu-
cleus. The strategies to target the NPC differ among viruses but invari-
ably involve specific pathways of signaling, endocytosis, access to the
cytosol, and cytoplasmic transport (for reviews, see Greber 2002; Meier
and Greber 2003; Ploubidou and Way 2001; Poranen et al. 2002; Sodeik
2000). Nuclear import of incoming viral genomes also depends on viral
uncoating and in some cases involves an increase of capsid affinity for
the NPC (for reviews, see Greber et al. 1994; Whittaker et al. 2000). In

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U.F. Greber • M. Fornerod

addition, certain viruses and viruslike particles (VLPs) undergo endoge-
nous replication and can shuttle their genomes in and out of the nucleus
without going through a complete virus assembly process.

6.1
Parvoviruses

The nonenveloped parvoviruses are among the smallest virus particles
found both intracellularly and extracellularly. They consist of a simple
icosahedral capsid of about 26 nm bearing a single-strand DNA genome
of about 5,000 bases. Parvoviruses internalize by receptor-mediated en-
docytosis, arrive in the cytosol, interact with the NPC, and deliver their
single-strand DNA into the nucleus, where the synthesis of the viral sec-
ond-strand DNA occurs (reviewed in Greber 2002; Sieczkarski and
Whittaker 2002). For example, the canine parvoviruses are small enough
to pass through the NPC without prior dissociation. However, microin-
jected cytosolic particles have not been found to be imported in signifi-
cant amounts into the nucleus (Weichert et al. 1998). In infected cells,
the amino-terminal sequence of the capsid protein VP1 is exposed by an
unknown stimulus, and this was found to correlate with an increased
nuclear localization of capsids or capsid fragments ( Vihinen-Ranta et al.
2002). Accordingly, genetic deletion studies with the minute virus of
mice (MVM) have shown that the amino-terminal basic clusters of VP1
are required for the onset of infection, suggesting that importin a and ji
are involved in VP1 import (Lombardo et al. 2002). It is not clear, how-
ever, whether additional NLSs on other capsid proteins need to be ex-
posed for nuclear import of the viral genome or whether the capsid dis-
assembles before DNA import (for further discussion, see Greber and
Fassati 2003).

Similarly, it is unclear how the adeno-associated viruses (AAVs) un-
coat and import their ssDNA genomes. One report states that the AAV
serotype 2 entered the nuclei in intact form (Bartlett et al. 2000). AAV2
was also reported to be imported into purified nuclei from cultured cells
in a nonsaturable manner, independent of temperature and NPC in-
hibitors (Hansen et al. 2001). Nuclei of parallel samples excluded the
150-kDa dextran but not the 4-kDa dextran, suggesting that they were
size selective and competent to retain small dextran. Another study,
however, suggested that fluorescent AAV2 rapidly entered HeLa cells and
remained in a perinuclear area up to 24 h after infection (Xiao et al.
2002). Viral uncoating was thought to happen before or during nuclear
entry of viral DNA and intact AAV particles were difficult to detect in-

Nuclear Import in Viral Infections

119

side the nuclei. In the presence of coinfecting Ad5, however, viral capsid
fluorescence was found within the nuclei and viral DNA cofractionated
with enriched nuclei. This was independent of the NPC inhibitor thapsi-
gargin (Greber and Gerace 1995), which blocked nuclear access of small
dextrans and AAV replication (Xiao et al. 2002). The data suggested that
in the presence of Ad5, AAV2 particles could enter the nucleus. The un-
derlying mechanisms of the proposed NPC-independent nuclear trans-
location are, however, unknown, and the nuclear factors triggering cap-
sid disassembly remain to be identified.

6.2

Hepadnaviruses, DNA Retroviruses

Hepadnaviruses, such as hepatitis B virus (HBV), are another family of
small viruses that could translocate through the NPC without disassem-
bly, just based on capsid size. Hepadnaviruses are enveloped viruses
with an icosahedral capsid of up to 36 nm and a partly double-strand
DNA genome that arises through reverse transcription from a prege-
nomic positive-sense RNA (Ganem and Schneider 2001). Human HBVs
are important pathogens, causing acute and chronic hepatitis and even-
tually liver cancer. Their entry is difficult to study in cell cultures for
reasons that are not precisely known. Recombinant hepatitis B virus
capsids produced in E. coli were found to associate with the NPCs of
permeabilized cells in the presence of importin a and /?, provided that
the capsids were phosphorylated by protein kinase C before incubation
with the permeabilized cells (Kann et al. 1999). PKC somehow exposed
an NLS in the carboxy terminus of the capsid proteins, and this seemed
to be important for the interaction with the NPC. A homologous NLS is
present in the duck hepatitis B virus (dHBV) capsid protein, but it is po-
sitioned more centrally in the protein and not near potential phosphory-
lation sites (Mabit et al. 2001). The dHBV NLS was essential for NPC as-
sociation of the nucleocapsid and for virus production as shown with a
knock-out mutant, confirming the important role of this NLS in the viral
life cycle. High-resolution EM in Xenopus oocytes subsequently showed
that recombinant HBV capsids that were phosphorylated with PKC were
localized at and within the NPCs but not in the nucleoplasm, suggesting
that the recombinant capsids were arrested in the NPC nuclear baskets
(Pante and Kann 2002). Because the baskets are known to be dynamic
and to have a high turnover (Daigle et al. 2001; Jarnik and Aebi 1991;
Nakielny et al. 1999), they might impose a diffusional barrier. Alterna-
tively, some NPC proteins might remain bound to these capsids through

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U.F. Greber • M. Fornerod

multiple direct and indirect interactions. A recent study suggests that
mature HBV capsids isolated from purified virions disassemble and re-
lease their DNA within the nucleus of permeabilized cells (Rabe et al.
2003). Surprisingly, the presence of the soluble factors importin a and (3
was sufficient to release a significant amount of DNA into the nucleus of
these permeabilized cells. Interestingly, immature viral capsids derived
from virion-producing hepatoma cell lines were found to be phosphory-
lated and bound to the NPC much like the E. coli- expressed capsids.
However, the immature capsids did not uncoat their nucleic acids which
were present as a mixture of RNA and DNA intermediates. This revealed
two critical steps for nuclear import of HBV DNA genomes. The first
step is the exposure of a capsid NLS, making the particle competent to
bind importins and associate with the NPC, and the second step is the
release of the reverse-transcribed double-strand DNA genome from the
capsid. Analogous to the disassembly program of the species C Ads and
herpes simplex virus type 1 (HSV1) (Greber 1998; Greber and Fassati
2003), HBV may contain a built-in priming mechanism that allows a dis-
assembly-competent capsid to associate with the critical cellular struc-
ture, the NPC, and there receive the final trigger for DNA release. The
nature of the final cue triggering disassembly of HBV capsids is still un-
known.

6.3
Retrotransposons

Retrotransposons containing long terminal repeats (LTR) are found in
many eukaryotic cells. They are closely related to retroviruses and en-
code Gag, reverse transcriptase (RT), and integrase (IN) proteins (Boeke
and Stoye, 1997). The absence of envelope (ENV) genes suggests that
they do not switch their host cells as frequently as the ENV- containing
retroviruses. Nonetheless, retrotransposons shuttle between the cyto-
plasm and the nucleus. Their Gag proteins assemble into coats, and this
is required for reverse transcription of the mRNA genome. IN integrates
the DNA genome into the host chromosomes. The maturation of the vi-
ral particles leads to Gag processing by the viral protease (PR), yielding
three major products, matrix (MA), capsid (CA), and nucleocapsid
(NC). For example, the retrotransposon Tyl and Ty2 of yeast Saccharo-
myces cerevisiae, the Tfl and Tf2 elements of Schizosaccharomyces
pombe, and the copia elements of Drosophila melanogaster form VLPs.
Tfl and Tf2 are model systems to study retroviruses (Levin et al. 1990),
and the expression of Tfl mRNA leads to high levels of transposition. A

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Gag-like protein has been shown to cosediment with a complex of RT,
IN, and viral cDNA (Levin et al. 1993). This Gag-like protein has an NLS
required for nuclear import and transposition (Dang and Levin 2000).
Nuclear import depends on the FXFG-type NPC protein Nupl24, which
is directly targeted by Tfl (Balasundaram et al. 1999; Dang and Levin
2000). Yeast cells mutated in Nupl24 block the nuclear import of Tfl
protein and also cDNA. This import defect appears to be specific, be-
cause overall protein or RNA import was not affected in this mutant. It
is not clear whether Nupl24 has a functional homolog in other eukary-
otes. Nupl24 shows the highest resemblance to S. cerevisiae Nuplp, a
nucleoporin located at the nuclear basket, but Nupl is essential. A nucle-
ar targeting signal in integrase is involved in nuclear transport of Ty3, a
retrotransposon of S. cerevisiae (Lin et al. 2001). It is not clear, though,
whether VLPs disassemble to let the retrotransposon genome cross the
NPC.

6.4

Lentiviruses

Like retrotransposons, the genomes of retroviruses are unspliced mRNA
that is packaged into a nucleocapsid. Unlike retrotransposons, the nucle-
ocapsid is enwrapped by a lipid membrane. The viral replication cycle
involves a double- strand DNA derived from the genomic mRNA by re-
verse transcription in the cytoplasm in the so-called reverse transcrip-
tion complex (RTC), giving rise to the preintegration complex (PIC), fol-
lowed by integration of the proviral DNA into the host chromosomes
(Cooper et al. 1995; for a recent perspective, see Trono 2003). Whereas
oncoretroviruses require dividing cells for infection, lentiviruses, such
as the human immunodeficiency virus (HIV), infect both dividing and
nondividing cells. The lentivirus PIC traffics through the cytoplasm and
is imported into the nucleus through the NPC (for recent reviews, see
Greber and Fassati 2003; Piller et al. 2003). Lentiviral DNA could be
found inside the nucleus of CD4 receptor- and CCR5 coreceptor-ex-
pressing HeLa cells (Bell et al. 2001). Large amounts of viral DNA were
not integrated into chromosomes, supporting the notion that these cells
had efficiently imported PIC. PIC is a pleiomorphic structure of double-
strand DNA and several viral proteins, including RT, IN, MA, and the
auxiliary protein Vpr as well as other unidentified cellular proteins
(Miller et al. 1997). RT is, however, found to be shed from the reverse
transcription complex soon after entry (Fassati and Goff 2001). Lentivi-
ral PIC also contains a particular structural DNA element, a polypurine

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tract serving as a second site of plus-strand DNA synthesis. Viral mu-
tants bearing deletions of this DNA flap structure have a slightly reduced
ability to access the nucleus (Zennou et al. 2000). Although IN and MA
contain basic NLSs that function in the context of the isolated proteins,
their roles in PIC import are controversial (this has been reviewed else-
where; see, e.g., Goff 2001; Greber and Fassati 2003; Piller et al. 2003).

The small accessory protein Vpr of 14 kDa plays several interesting
roles. It appears to enhance HIV infection in macrophages, and, interest-
ingly, it interacts with the NPC (Fouchier et al. 1998; Popov et al. 1998;
Vodicka et al. 1998). However, Vpr is not essential for viral infectivity in
all nondividing cells; for example, it is not needed in T cells (Eckstein et
al. 2001). The role of Vpr in nuclear import of PIC has been controver-
sial (see, e.g., Bouyac-Bertoia et al. 2001). Nonetheless, Vpr uses several
nuclear import pathways that are Ran- and importin independent
(Jenkins et al. 1998). Moreover, it shuttles between the nucleus and the
cytoplasm through a CRM1 -dependent NES (Sherman et al. 2001). Fur-
thermore, yeast two-hybrid interactions, in situ localizations, and coim-
munoprecipitations have shown that Vpr associates with the NPC pro-
tein hCGl, the mammalian homolog of the yeast nucleoporin Riplp/
Nup42 (Le Rouzic et al. 2002). hCGl was originally identified in yeast
two-hybrid screens binding to the human export factor TAP and to
CRM1 binding the HIV-1 Rev protein (Farjot et al. 1999; Katahira et al.
1999; Wiegand et al. 2002). The yeast homolog Riplp is involved in the
export of mRNAs encoding heat shock proteins (Saavedra et al. 1997),
and a carboxy-terminal truncation of Riplp can be rescued by the car-
boxy terminus of hCGl, suggesting that Riplp and hCGl are performing
homologous functions (Strahm et al. 1999). Vpr interacts with FG re-
peats of several nucleoporins in vitro, but it does not use the FG repeats
of hCGl and rather binds a nonconserved domain in the amino-termi-
nal portion of hCGl (Le Rouzic et al. 2002). In contrast, the hCGl FG
repeats interact with the transport factors TAP and CRM1, implying that
Vpr might not interfere with hCGl -dependent export pathways. The
functional implications of the Vpr interactions with hCGl are, however,
not known. It is also unknown whether Vpr disturbs normal NPC trans-
port functions and how it is involved in the formation of herniations
and transient perforations of the nuclear envelope and cell cycle arrest
in the G 2 phase (de Noronha et al. 2001). Nevertheless, it is conceivable
that, depending on the cell type and the viral isolate, the association of
Vpr with the NPC is a key event supporting PIC import and infection.

Another class of key factors of RTC nuclear import are likely to be
the importins. A recent analysis of transport factors of RTC import in

Nuclear Import in Viral Infections

123

primary macrophages points toward importin 7, which is normally in-
volved in histone HI import (Fassati et al. 2003). Using in vitro nuclear
import assays in permeabilized cells and small interfering RNAs for im-
portin 7, these authors showed that importin 7 stimulated nuclear im-
port of RTC in a Ran-dependent manner and was required in HIV infec-
tion. It was preferentially involved in IN import, but IN interacted with
other importins, including importin (5. The inhibition of the importin j3
pathway by an excess of the importin j5 binding domain had only mod-
erate effects on RTC import, suggesting that importin (5 is not sufficient
for RTC import (Gallay et al. 1997). Consistently, mutations of basic
NLSs in IN did not affect nuclear import of PIC and had little effect on
IN import into HeLa cells (Petit et al. 2000). However, IN contains an-
other nonbasic NLS located in the middle of the protein. This NLS is re-
quired for accumulation of viral DNA in the nucleus and for infection
but does not affect the catalytic activity of IN (Bouyac-Bertoia et al.
2001). It will now be important to develop a coherent picture of how the
different players of PIC import are coordinated and how their action re-
lates to the conformation of the PIC and its precursor, the RTC. Interest-
ingly, some consensus of RTC structure and function now seems to
emerge based on microscopy and biochemical analyses. It is suggested
that after viral fusion with the plasma membrane the incoming lentiviral
core remains intact until it is delivered into the cytosol (Fassati and Goff
2001; McDonald et al. 2002; Nermut and Fassati 2003). Reverse tran-
scription and additional cues then induce capsid uncoating, and this
might in turn be essential for the completion of the reverse transcription
process. Intriguingly, host restrictions targeting the p24 Gag component
CA might help inhibiting viral uncoating (Besnier et al. 2002), and thus
viral uncoating and subsequent import can be points of host antiviral
defense (Turelli et al. 2001). Resolving the molecular mechanisms under-
lying these blocks promises to reveal more of the intricate relationship
of retroviruses with their hosts.

6.5

Adenoviruses

Adenoviruses (Ads) are large nonenveloped DNA viruses with a capsid
diameter of about 90 nm (Burnett 1997). Their entry pathways are large-
ly determined by the fiber proteins that bind particular cell surface re-
ceptors. Ad particles navigate their genome through the cytoplasm along
microtubules, much like the HSV1 capsids (Mabit et al. 2002). In con-
trast to retroviral capsids, the Ad capsids dock to the NPC of both in-

124

U.F. Greber • M. Fornerod

fected cells and purified nuclei (Chardonnet and Dales 1970; Morgan et
al. 1969; Wisnivesky et al. 1999). The species C Ads dock to a nucleo-
porin, CAN/Nup214 of the cytoplasmic side of the NPC in the absence
of cytosolic factors (Trotman et al. 2001). In all likelihood, docking oc-
curs through the major capsid protein hexon. However, docking is not
sufficient for DNA release from the capsid, which is crucial for infection
(Cotten and Weber 1995; Greber et al. 1997, 1996). To uncoat, the NPC-
docked Ad2 particle binds the mobile linker histone HI that exits the
nucleus for short periods (Trotman et al. 2001). This binding is thought
to occur at the acidic polyglutamate domains of hexon located at the
outside of the capsid (Rux and Burnett 2000). These acidic stretches are
conserved in the species C Ads but not in other species. Binding of HI
is, however, still not sufficient for Ad2 disassembly, and the histone HI
import factors must be present (Trotman et al. 2001). Additional cyto-
solic factors such as Hsp70 have been implicated as well (Saphire et al.
2000). It is unknown how these molecular requirements relate to the ear-
lier notion that cells treated with p-hydroxymercuric benzoate (PHMB)
block viral uncoating and inhibit a cellular ATPase activity present in
isolated nuclei (Chardonnet and Dales 1972; Dales and Chardonnet
1973). It should be noted here that the Ad2 capsids that are docked to
the NPCs have previously gone through a limited disassembly program,
removing the fibers and destabilizing minor capsid components (Greber
et al. 1993; Nakano et al. 2000). Thus it is presumably the simultaneous
binding of the carboxy- terminal domain of CAN/Nup214, the histone
HI, and the HI import factors importin j3 and importin 7 on the weak-
ened capsid that finally triggers capsid disassembly, allowing the release
of the viral DNA. How Ad serotypes of species other than species C dock
to the NPC and uncoat their genomes is unknown.

6.6
Herpesviruses

Most of the information about nuclear import of herpesviruses comes
from studies with herpes simplex virus type 1 (HSV1) and pseudorabies
virus (PRV), belonging to the alpha herpesviruses subfamily. After fusion
of the viral envelope with the plasma membrane, the 120-nm large HSV1
and PRV nucleocapsids are transported through the cytoplasm and are
targeted to the NPCs similar to Ad capsids, as indicated by EM of cul-
tured cells (Granzow et al. 1997; Lycke et al. 1988; Roizman and Knipe
2001; Sodeik et al. 1997). Remarkably, the capsid seems to be positioned
in a particular orientation with respect to the NPC, perhaps placing the

Nuclear Import in Viral Infections

125

portal protein channel, through which the viral DNA was packaged, prox-
imal to the NPC mouth (Newcomb et al. 2001). The docked capsids un-
coat and inject their linear double-strand DNA genome through the nu-
clear pores into the nucleoplasm and often leave an empty capsid behind
at the NPC (for a recent review, see Greber and Fassati 2003). The precise
composition of the NPC-docked capsids is not known, but the capsids
comprise at least four different proteins and additional factors from the
tegument — a mass of protein and mRNA located between the nucleocap-
sid and the envelope in intact virions. Interestingly, the HSV-1 mutant
tsB7 appears to be defective at capsid disassembly and/or DNA release if
grown at the restrictive temperature but this mutant still docks at the
NPC (Knipe and Smith 1986). Although tsB7 bears multiple mutations,
this may mean that tegument proteins are part of a viral checkpoint for
DNA release. Indeed, capsids containing the major tegument proteins
VP1/2, VP13/14, and VP22 were found to bind to isolated nuclei when re-
combinant importin j3 was present (Ojala et al. 2000). Capsid binding to
nuclei was inhibited by an excess of Ran:GTP, implying that importin j5
bound to capsids. The addition of cytosol and ATP together with shifting
the temperature up to 37°C triggered genome uncoating, that is, the viral
DNA became sensitive to added deoxyribonuclease. Although the cyto-
solic factors required for genome uncoating are unknown, the removal of
the tegument proteins VP1/2, VP13/14, and VP22 by proteolytic diges-
tions before nuclear binding inhibited capsid binding to nuclei and DNA
release in vitro. Studies of viral mutants, furthermore, suggested that the
full-length VP22 is not needed for a single round of HSV1 infection, in-
cluding viral entry, replication, and assembly, but that it may be required
for spreading virus particles in vivo (reviewed in Mettenleiter 2002). This
implies that VP22 may not be a key factor of HSV capsid docking to the
NPC. It will be interesting to see whether HSV capsids release their DNA
at a specialized region in a capsid vertex, for example, the portal of entry,
which is known to be important for the packaging of the genome into
the newly synthesized capsids (for a discussion, see Fuller 2003).

6.7

Papovaviruses

The papilloma and polyoma virus subfamilies (papovavirus family)
comprise more than 80 different human and animal viruses (Cole 1996;
Howley 1996). They are nonenveloped icosahedral double-strand DNA
viruses about 60 nm in diameter. Depending on the virus and the cell
type, their entry occurs by clathrin-mediated endocytosis, caveolar en-

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U.F. Greber • M. Fornerod

docytosis, or noncaveolar cholesterol-dependent endocytosis (Ashok
and Atwood 2003; Bousarghin et al. 2003; Day et al. 2003; Fausch et al.
2003; Pelkmans et al. 2001; Pho et al. 2000; Selinka et al. 2002). In some
cases entry also involves coreceptors (for a recent example, see Caruso
et al. 2003).

One of the better-studied polyoma viruses is the simian vacuolating
virus 40 (SV40). SV40 entry occurs via cholesterol-rich membrane do-
mains that are enriched in signaling molecules (reviewed by Pelkmans
and Helenius 2002). In caveosomes, SV40 is sorted for transport to the
smooth ER from where it somehow transfers its DNA into the nucleus.
This seems to involve a cytosolic phase, because neutralizing anti-SV40
antibodies injected into the cytoplasm inhibited the infection (for re-
views of the older literature, see Greber and Kasamatsu 1996; Kasamatsu
and Nakanishi 1998). There is recent evidence to suggest that SV40 ex-
poses the NLS of the minor VP2/3 protein and this NLS is recognized by
importin a (Nakanishi et al. 2002). Some of the key questions that re-
main to be solved include Where do these conformational changes occur
in the cell? and What is the composition of the nucleoprotein complex
that is delivered into the nucleus?

Papillomaviruses are more difficult to study because the infectious
units are relatively rare in isolated virions and the viruses replicate in
terminally differentiated keratinocytes (Howley and Lowy 2001). The
high-risk human papillomaviruses (including HPVs 16, 18, 31, 33, 45)
are frequently found in invasive cervical carcinomas, whereas the low-
risk HPVs (types 6 and 11) are associated with rather benign condylo-
mata acuminata (zur Hausen 2002). Nuclear import of HPVs has been
studied with viruslike particles (VLPs) and the capsid proteins LI and
L2. The LI protein is the major capsid component and forms VLPs in
the absence of L2, but incorporation of L2 seems to be important for in-
fectivity (Florin et al. 2002). Intact LI capsids of HPV11 were found to
be excluded from the nucleus, but LI capsomeres were imported into
the nuclei of digitonin-permeabilized HeLa cells (Merle et al. 1999). In
this system, docking of HPV11 VLPs was inhibited by antibodies against
importin al and /Jl. These results suggested that capsid disassembly
was required for HPV11 LI nuclear import and that the classic im-
portins and additional factors were involved. Likewise, the LI protein of
the high-risk HP VI 6 was imported into the nucleus of digitonin-perme-
abilized HeLa cells in a complex with importin al and j3\ in a Ran-de-
pendent manner (Nelson et al. 2002). Interestingly, it was observed in
this study that LI capsomeres also interacted with importin (31, which
is normally involved in the import of M9- containing proteins, such as

Nuclear Import in Viral Infections

127

hnRNP Al. The related LI of the low-risk HPV11 also interacted with
importin (32 and importin /?3 and thereby inhibited the nuclear import
of hnRNP Al (Nelson et al. 2003). Whether this has any physiological
significance during virus assembly or release is unknown.

6.8

Negative-Sense RNA Viruses

Influenza virus is an orthomyxovirus with a segmented negative-sense
RNA genome that requires the nucleus for replication (reviewed in Lamb
and Krug 2001). It contains a lipid-protein envelope and eight different
genomic RNPs containing the RNA, nucleoprotein (NP), and the het-
erotrimeric RNA-dependent RNA polymerase. The RNPs are tethered to
the envelope through the matrix protein M. Although the length of the
RNPs varies from about 20 to 100 nm, their diameter is probably smaller
than 25 nm, that is, it would pass through the NPC without major un-
coating reactions. To a large extent, viral disassembly happens in late en-
dosomes, where the low pH triggers conformational changes of viral he-
magglutinin, which then acts to catalyze fusion of the viral envelope
with the limiting endosomal membrane (for a recent review, see Colman
and Lawrence 2003). In addition, endosomal protons dissociate the in-
teractions of the matrix protein Ml and the RNPs within the virion,
which is required for NP nuclear import in cultured cells (reviewed in
Lamb et al. 1994; Whittaker et al. 2000). Import of RNPs has also been
investigated in digitonin-permeabilized cells. It was found that RNA im-
port required the coating of the RNA with NP and the presence of im-
portin a (i.e., the npi-1 and npi-3 proteins), importin j3 and Ran (O'Neill
et al. 1995; Wang et al. 1997). The NLS on NP is nontypical, namely,
SxGTKRSYxxM for npi-1 and TKRSxxxM for npi-3, different from the
basic SV40 large T-type NLS. Additional predominantly basic NLSs have
been identified in the NP protein, but it is not clear whether, in the con-
text of the RNP, these NLSs bind the nucleic acid or import factors.

Thogoto virus, a tick-borne orthomyxovirus with six genomic seg-
ments of negative polarity and a structure much like influenza virus,
provides an interesting example where nuclear trafficking of the incom-
ing viral genome is intercepted by an innate host defense mechanism
based on the interferon-induced large GTPases of the Mx family (for a
recent review, see Haller and Kochs 2002). Mx proteins were shown to
bind the viral RNPs, and this inhibited RNP nuclear import (Kochs and
Haller 1999). Yet another example of Mx protein-mediated interference
with cytoplasmic trafficking of viral RNPs has been reported with bun-

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U.F. Greber • M. Fornerod

yaviruses, namely, La Crosse virus (LACV), an arbovirus causing pediat-
ric encephalitis (Kochs et al. 2002). LACV replicates in the cytoplasm,
but in MxA expressing cells the LACV RNPs were sequestered into cyto-
plasmic fibrillar structures and rendered unavailable for replication. It
will be interesting to see whether the cytoplasmically sequestered gen-
omes are targeted for degradation or whether the cell enters a self de-
struction program before producing progeny virus.

Perspectives

The ongoing molecular analysis of viral import into the nucleus has re-
vealed that incoming viruses undergo several conformational changes
that allow them to destabilize the protective capsid and in some cases
increase their nuclear affinity. In the case of DNA and RNA retroviruses,
some of these changes are linked to genome maturation processes in the
cytoplasm, rendering the capsids competent for cytoplasmic transport
and interactions with the nuclear pore complexes. Viral nucleoproteins
interact with the NPC both directly and through cellular import factors.
Direct attachment to the NPC is particularly effective in the case of
viruses that have a relatively hydrophobic surface such as the Ads or the
parvoviruses. These direct interactions can serve to position the particle
such that it becomes competent for releasing the nucleic acid. Alterna-
tively, direct interactions can lead to capsid translocation through the
pore, resulting in infection after intranuclear genome uncoating. Impor-
tant challenges for future research include the identification of the nu-
clear, cytoplasmic, and viral factors required for genome uncoating,
identification of the factors involved in NPC translocation of the nucleo-
protein complexes, and tackling the subnuclear events immediately after
nuclear import. We expect that research on nuclear import of incoming
viral particles will continue to reveal basic cellular mechanisms, which
might include new aspects of the innate host antiviral defense, similar to
antiviral signaling processes.

Acknowledgements Research in the lab of UFG is supported by funds from the Swiss
National Science Foundation, the Cancer League of the Canton of Zurich, and
the Canton of Zurich. MF is supported by a grant from The Netherlands Cancer
Institute.

Nuclear Import in Viral Infections

129

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© Springer- Verlag 2004

Viral RNA Replication in Association
with Cellular Membranes

A. Salonen • T. Ahola • L. Kaariainen ([*])

Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter,
University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland
leevi.kaariainen@helsinki.fi

1 Introduction 140

2 Alphaviruses as Models 141

2.1 RNA Replication in Cytoplasmic Vacuoles Derived from Endosomes . . 141

2.2 nsPl as the Membrane Anchor of the Replication Complex 143

2.3 Membrane Binding Mechanism of nsPl 144

2.4 Polyprotein Conducts the Assembly and Targeting

of the Replication Complex 147

3 Alphavirus-Like Superfamily 150

4 Picornavirus-Like Superfamily 154

4.1 Poliovirus as a Model 154

4.2 Other Members of the Picornavirus Superfamily 158

5 Flavivirus-Like Superfamily 159

6 Nidoviruses 162

7 Concluding Remarks 163

7.1 Targeting of the Replicase Complex 163

7.2 Mechanisms of Membrane Binding 164

7.3 Membrane Modification by Replicase Proteins 164

7.4 Functional Implications of Membrane Attachment 167

References

167

Abstract All plus-strand RNA viruses replicate in association with cytoplasmic
membranes of infected cells. The RNA replication complex of many virus families is
associated with the endoplasmic reticulum membranes, for example, picorna-, flavi-,
arteri-, and bromoviruses. However, endosomes and lysosomes (togaviruses), perox-
isomes and chloroplasts (tombusviruses), and mitochondria (nodaviruses) are also
used as sites for RNA replication. Studies of individual nonstructural proteins, the
virus-specific components of the RNA replicase, have revealed that the replication
complexes are associated with the membranes and targeted to the respective orga-
nelle by the ns proteins rather than RNA. Many ns proteins have hydrophobic se-
quences and may transverse the membrane like polytopic integral membrane pro-

140

A. Salonen et al.

teins, whereas others interact with membranes monotopically. Hepatitis C virus ns
proteins offer examples of polytopic transmembrane proteins (NS2, NS4B), a "tip-
anchored" protein attached to the membrane by an amphipathic a-helix (NS5A) and
a "tail-anchored" posttranslationally inserted protein (NS5B). Semliki Forest virus
nsPl is attached to the plasma membrane by a specific binding peptide in the middle
of the protein, which forms an amphipathic a-helix. Interaction of nsPl with mem-
brane lipids is essential for its capping enzyme activities. The other soluble replicase
proteins are directed to the endo-lysosomal membranes only as part of the initial
polyprotein. Poliovirus ns proteins utilize endoplasmic reticulum membranes from
which vesicles are released in COPII coats. However, these vesicles are not directed
to the normal secretory pathway, but accumulate in the cytoplasm. In many cases
the replicase proteins induce membrane invaginations or vesicles, which function as
protective environments for RNA replication.

Introduction

The genome replication of all plus-strand RNA viruses infecting eukary-
otic cells is associated with cellular membranes. The membranes can be
derived from the endoplasmic reticulum (ER), or other organelles of the
secretory pathway, mitochondria, chloroplasts, or from the endo-lyso-
somal compartment. The membrane association provides a structural
framework for replication, it fixes the RNA replication process to a spa-
tially confined place, increasing the local concentration of necessary
components, and it offers protection for the alien RNA molecules
against host defense mechanisms. The theme of immobilized polymer-
ase and moving template may in fact be common also to most cellular
DNA replication and transcription systems (Cook 1999) and might
therefore reflect a primordial pathway in nucleic acid replication. The
modes of membrane binding and targeting to specific intracellular or-
ganelles of the replication complexes of different viruses are so far poor-
ly understood. However, this field at the interface of virology, cell biolo-
gy, and biochemistry is attracting increased interest, as it represents an
ancient feature shared by many virus groups. Some aspects have been
nicely treated in earlier reviews (de Graaff and Jaspars 1994; Buck 1996).
We will present in some detail relevant studies on alphaviruses, especial-
ly Semliki Forest virus (SFV), which has been the object of our own in-
terest. We will then review recent work on other viruses based on their
classification in three superfamilies (Koonin and Dolja 1993), except that
we have treated nidoviruses as a separate group.

Viral RNA Replication in Association with Cellular Membranes 141

Alphaviruses as Models
2.1

RNA Replication in Cytoplasmic Vacuoles Derived from Endosomes

Association of SFV-specific RNA synthesis with membranes was demon-
strated in several studies starting in the late 1960s (for reviews see
Kaariainen and Soderlund 1978; Kaariainen and Ahola 2002). Simple
fractionation of membranes derived from the postnuclear supernatant
fraction of alphavirus-infected cells showed that essentially all RNA
polymerase activity was associated with a "mitochondrial" pellet frac-
tion sedimenting at 15,000 x g. On the other hand, early electron micro-
scopic (EM) studies had revealed cytoplasmic structures typical for al-
phavirus-infected cells. These were designated as "cytopathic vacuoles
type I" (CPV-I), hereafter referred as CPVs. Their size varied from
600 nm to 2,000 nm, and their surface consisted of small vesicular invag-
inations or spherules, of homogenous size, with a diameter of about
50 nm. EM autoradiography of SFV- infected cells pulse-labeled with tri-
tiated uridine already suggested that CPVs, and possibly the spherules,
were involved in virus-specific, actinomycin D-resistant RNA synthesis
(Grimley et al. 1968).

However, the origin, nature, and function of CPVs remained unclear
for about two decades until Froshauer et al. (1988) demonstrated that
they were modified endosomes and lysosomes. Immunofluorescence
and immuno-EM techniques showed that Sindbis virus-specific non-
structural proteins nsP3 and nsP4 were associated with CPVs. The au-
thors proposed that CPVs were derived from endosomes, which were
participating in the internalization of virus particles. This would have
nicely explained the endosomal origin of CPVs as a direct consequence
of fusion of the virus envelope with the endo/lysosomal membrane,
which would bring the virus nucleocapsid and genome directly to the
cytoplasmic surface of the organelle. Thus genome uncoating and subse-
quent synthesis of replicase components would result in the modifica-
tion of endosomes to virus-specific CPVs. However, this hypothesis can-
not explain why the amount of CPVs was not dependent on the amount
of infecting virions. Moreover, typical CPVs were seen also in cells trans-
fected with the genomic RNA of SFV, demonstrating that the endosomal
targeting of the replication complexes must be a posttranslational event
(Peranen and Kaariainen 1991).

142

A. Salonen et al

Cap

nsP1

nsP2

nsP3 nsP4

^ Translation

T Processing

P123

nsP4j>

Minus strand Pol

nsP3

nsP1

nsP2

nsP4

Plus strand Pol

Palmitoylation C418-C420

nsP1

Conserved domain

245 " ^ 264

GSTLYTESRKLLRSWHLPSV

537

Fig. 1. A Genome organization of SFV. The translation and processing products rele-
vant for SFV replication are shown. Physical interactions have been identified be-
tween nsPl and nsP4, as well as nsPl and nsP3 (Salonen et al. 2003). B Scheme of
SFV nsPl showing the two regions responsible for membrane binding. The amino
acid sequence of the lipid binding peptide is given in single-letter code, and the posi-
tion of palmitoylated cysteines is marked

Alphavirus nonstructural (=replicase) proteins are synthesized as a
polyprotein precursor PI 234, which is processed in a highly regulated
manner into the individual components nsPl-nsP4 (Fig. 1A). Genetic
and biochemical experiments have revealed many of the functions of the
nsPs (reviewed in Strauss and Strauss 1994; Kaariainen and Ahola 2002).
Thus nsP4 is the catalytic RNA-dependent RNA polymerase subunit,
nsP2 is involved in the regulation of the synthesis of the subgenomic 26S
mRNA coding for structural proteins of the virion, whereas nsPl is
needed in the synthesis of the complementary (minus strand) RNA early
in infection. nsP3 is essential for infection, but no specific function has
been assigned for it as yet. Expression of the individual nsPs in E. coli
and in insect cells revealed further functions of nsPs. nsPl is an RNA
capping enzyme with unique methyltransferase and guanylyltransferase
activities (Mi and Stollar 1991; Ahola and Kaariainen 1995), whereas
nsP2 turned out to be a NTPase and RNA helicase (Gomez de Cedron at

Viral RNA Replication in Association with Cellular Membranes 143

al. 1999), RNA triphosphatase (Vasiljeva et al. 2000), as well as the prote-
ase responsible for the processing of the nonstructural polyprotein pre-
cursor (Vasiljeva et al. 2001).

Creation of potent monospecific antibodies allowed the identification
and localization of the ns proteins in SFV-infected cells (Peranen et al.
1988; 1995). After crude cell fractionation most of nsPl, nsP3, and nsP4
were associated with the P15 membrane fraction, whereas about 50% of
nsP2 was found in the nucleus (Peranen et al. 1990). Pairwise double
staining with different anti-nsP antibodies revealed costaining of CPV-
like structures in immunofluorescence microscopy, suggesting that all
four nsPs were associated with CPVs. This was confirmed by double-la-
beling in cryo-immuno EM (Kujala et al. 2001). Moreover, bromouridine
given in short pulses also localized to CPVs and spherules together with
the nsPs, indicating that these structures were the sites of RNA replica-
tion. The CPVs costained with late endosomal (lamp-1, lamp-2, and
rab7) and lysosomal markers (LBPA and LysoTracker). Interestingly, all
nsPs had localization sites also outside of the CPVs. nsP2 was found in
the nucleus, nsPl at the plasma membrane, nsP3 in cytoplasmic spotlike
structures, and nsP4 diffusely in the cytoplasm (Kujala et al. 2001).
Therefore, only a fraction of nsPs are present in the actual replication
complexes.

2.2

nsPl as the Membrane Anchor of the Replication Complex

As none of the alphavirus nsPs has sequences typical for transmem-
brane proteins, we have studied their membrane binding by expressing
them individually in BHK, HeLa, and insect cells. These studies revealed
that only nsPl had a specific association with membranes (Peranen et
al. 1995), whereas nsP2 on its own was transported almost quantitatively
to the nucleus and nsP3 was in cytoplasmic aggregates (Salonen et al.
2003), which in light microscopy gives an impression of vesicles of vari-
able size (Vihinen et al. 2001). Finally, nsP4 was distributed diffusely in
the cytoplasm.

Thus nsPl was a promising candidate as the membrane anchor of the
SFV replication complex. nsPl turned out to be very tightly membrane
bound, as the association was not sensitive to high salt, EDTA, or alka-
line sodium carbonate treatments, which release peripheral membrane
proteins (Peranen et al. 1995). The tight binding was due to palmitoyla-
tion of cysteine residues 418-420 (Fig. IB). When these residues were
mutated to alanines, nsPl was still membrane associated, but less tightly,

144

A. Salonen et al.

as it could now be released by high-salt treatment. Thus elimination of
palmitoylation altered nsPl from an "integral" to a "peripheral" mem-
brane protein (Laakkonen et al. 1996).

To study the significance of the palmitoylation of nsPl, the C418-
420A mutation was introduced to the infectious cDNA of SFV, followed
by transcription of genomic RNA, which was used for transfection of
BHK cells. Infectious virus was released to the medium, indicating that
palmitoylation of nsPl was not essential for virus replication. However,
there was some retardation in the kinetics of virus growth. Analysis of
the membrane association of wild-type and palmitoylation-negative mu-
tant (lpa-) replicase proteins of SFV showed that lpa- nsPl was bound
less tightly to the membranes than the wild-type protein. Typical CPVs
with spherules, indistinguishable from those in wild-type SFV-infected
cells, were seen in EM. The same results were obtained when the single
palmitoylated cysteine residue (C420) of Sindbis virus nsPl was mutated
to alanine. However, the SFV lpa- mutant was apathogenic for mouse.
After intraperitoneal infection blood viremia was detected, but no infec-
tious virus was found in the brain (Ahola et al. 2000).

2.3

Membrane Binding Mechanism of nsPl

Because palmitoylation was not the decisive mechanism for membrane
binding of nsPl, we studied the peripheral binding by producing the
wild-type protein in E. coli, which cannot palmitoylate proteins. Enzy-
matically active nsPl was associated with bacterial membranes as
judged by flotation in sucrose gradients. In vitro translated nsPl also as-
sociated with liposomes containing 20%-50% phosphatidylserine (PS)
or other anionic phospholipids, but not with liposomes containing only
phosphatidylcholine (PC). Solubilization of membranes containing nsPl
by detergents, such as Triton X-100 or octylglucoside, resulted in loss of
the protein's methyltransferase and guanylyltransferase activities, which
could be reactivated by reconstitution of nsPl into vesicle membranes
or into mixed detergent-lipid micelles containing anionic phospholipids
(Fig. 2A). Detergents also inhibited the binding of the methyl donor
S-adenosylmethionine to nsPl, and the binding was resumed under the
same conditions as enzymatic activity. Thus binding to anionic phos-
pholipids causes a conformational change, which activates the protein
(Ahola et al. 1999).

The membrane-binding site in nsPl was identified by site-directed
mutagenesis and deletion mapping in both bacterial and in vitro expres-

Viral RNA Replication in Association with Cellular Membranes

145

5 200
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Fig. 2. Biochemical characterization of the lipid binding of SFV nsPl. A Inactivation
and activation of nsPl methyltransferase activity. Triton X-100 inactivates nsPl,
compared with the control reaction in the absence of detergent (on the right). When
TX-100 micelles containing increasing amounts of phosphatidylserine are added,
nsPl regains activity, even exceeding control levels at optimal concentration of the
lipid. (Reproduced from Ahola et al. 1999, with permission). B Negatively charged
phospholipids increase the intensity of tryptophan fluorescence of the lipid binding
peptide. Tryptophan emission spectrum was recorded in the buffer or in the pres-
ence of small unilamellar vesicles consisting of phosphatidylcholine (PC) or PC with
30 mol % of phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidic acid
(PA), or phosphatidylethanolamine (PE). C Depth of tryptophan W259 penetration
to membrane measured by quenching of tryptophan fluorescence by brominated
PCs. The position of bromine in the acyl chains is indicated. D The peptide adopts
an a-helical conformation in the presence of PC-containing vesicles, as measured by
circular dichroism spectroscopy. The numbers indicate the mol % of PS in the vesi-
cles. (B-D reproduced from Lampio et al. 2000 with permission by the American
Society of Biochemistry and Molecular Biology)

146

A. Salonen et al.

sion systems. Flotation of nsPl with membranes or liposomes in discon-
tinuous sucrose gradients was used as a criterion for membrane associa-
tion. By this means a putative binding region of about 20 amino acid
residues, starting from Gly245, was identified (Fig. IB). The correspond-
ing synthetic peptide consisting of Gly245-Val264 (GSTLYTESRKLLR-
SWHLPSV) was able to compete with the binding of in vitro synthesized
nsPl to liposomes containing PS, strongly suggesting that this region of
nsPl is responsible for its membrane association (Ahola et al. 1999).

The interaction of the synthetic membrane binding peptide with lipo-
somes was assayed by utilizing the fluorescence of tryptophan residue
W259 (Lampio et al. 2000). Tryptophan emission spectrum changes
when it is embedded into an apolar environment. There was a marked
increase in the fluorescence intensity and a blue shift of the emission in
the presence of monolamellar liposomes, which consisted of PC and
negatively charged phospholipids (PS, phosphatidylglycerol, or phos-
phatide acid) (Fig. 2B). By using phospholipids with bromide substitu-
tion in different carbon atoms of the acyl chain, for quenching of the
tryptophan fluorescence, it was possible to estimate that W259 penetrat-
ed to the level of carbon atoms 9 and 10 of the PC acyl chains in the out-
er leaflet of the liposomes (Figs. 2C and 3A). The circular dichroism
spectrum of the binding peptide was dependent on the content of the
apolar constituents (liposomes or trifluoroethanol). In a buffer solution
the binding peptide was mostly in a random coil, whereas in the pres-
ence of liposomes with 20%-30% PS or in 30%-50% trifluoroethanol
the peptide attained an a-helical conformation (Fig. 2D). The solution
structure of the binding peptide, determined by NMR spectroscopy in
30% trifluoroethanol, revealed an amphipathic a-helix (Fig. 3A). One
face consisted of hydrophobic residues, leucines 248, 255, 256, and 261,
valine 264, and residues S252 and W259 interacting with the apolar fatty
acid chains on the cytoplasmic leaflet of the membrane (Fig. 3). The oth-
er face contained positively charged residues R253, K254, and R257 lying
parallel to the polar head groups of the bilayer surface. The hydrophobic
surface of the peptide is rather stable, whereas the polar residues show
considerable mobility in trifluoroethanol.

Point mutations R253E and W259A in the nsPl protein, when ex-
pressed in E. coli, resulted in the loss of enzymatic activity and the lack
of ability to float with membranes in sucrose gradients (Ahola et al.
1999). Thus these two residues were considered to be essential in the in-
teraction of nsPl with membranes. This view was supported by compe-
tition experiments done with synthetic mutant peptides. Both were un-
able to inhibit the binding of in vitro synthesized nsPl to liposomes, in

Viral RNA Replication in Association with Cellular Membranes

147

R253

W259

'iin

nsP1

Cytoplasm

Plasma membrane I ill |i

iftf i

Exterior

Fig. 3 A, B. Monotopic binding of nsPl to membrane via amphipathic a-helical pep-
tide. A The NMR structure of the peptide is shown in interaction with the cytoplas-
mic leaflet of the lipid bilayer. B Highly schematic overview of interaction of nsPl
with a lipid bilayer

contrast to the synthetic wild-type binding peptide (Lampio et al. 2000).
When the corresponding mutations were introduced to the SFV genome,
neither W259A nor R253E was able to produce infectious virus after
transfection (Salonen et al., unpublished data). Altogether, these results
indicate that the interaction of nsPl binding peptide with membranes is
an essential and structurally finely tuned process, dependent on interac-
tion with anionic phospholipids.

2.4

Polyprotein Conducts the Assembly and Targeting
of the Replication Complex

The nsPs are derived from a common precursor PI 234, the initial cleav-
age products of which (P123 plus nsP4) are necessary for the first step
in the RNA replication, the synthesis of complementary RNA (Lemm et
al. 1994, 1998) (Fig. 1A). To understand the role of this and other cleav-

148

A. Salonen et al.

age intermediates, we have produced them both in wild-type form and
as noncleavable polyprotein variants, in which the autoprotease of nsP2
was inactivated by a mutation of the active site cysteine to alanine
(superscript "CA"). The constructs were expressed in insect and mam-
malian cells, and the localization of individual proteins was followed by
confocal microscopy and the complex formation by immunoprecipita-
tion (Salonen et al. 2003).

The cleavable polyproteins (P12, P23, P123, P1234) containing an ac-
tive nsP2 protease were processed to their constituents, most of which
were distributed in the cell as though they were expressed alone. For in-
stance, P12 yielded nsPl and nsP2, which were targeted to the plasma
membrane and nucleus, respectively. However, flotation analysis and im-
munoprecipitation recapture experiments showed that expression of
P123 and P1234 resulted in membrane-bound complexes, containing all
the individual proteins. This was different from the coexpression of all
four nsPs individually, allowing us to conclude that the membrane asso-
ciation of the complex is guided by the polyprotein intermediate.

The uncleavable polyproteins were palmitoylated and had enzymatic
activities typical for nsPl and nsP2, and those containing nsP3 were
phosphorylated, suggesting that the individual domains had folded
properly in the context of the polyprotein. When P12 CA was expressed in
HeLa cells, it was localized exclusively at the cytoplasmic side of the
plasma membrane and in long filopodia-like extensions (Fig. 4A), indis-
tinguishable from those previously described for cells expressing nsPl
alone (Laakkonen et al. 1998). This indicated that the affinity to plasma
membrane of nsPl in the polyprotein overruled the attraction of nsP2
for nuclear transport. However, an interesting change in the localization
was seen when P12 CA 3 was expressed (Fig. 4B). No filopodia-like exten-
sions were seen, and instead intracellular vesicular staining was ob-
served, which in immuno-EM resembled CPVs (Fig. 4C and D). Double
immunofluorescence with antisera against nsPs and lamp-2 suggested
that at least a fraction of the vesicular structures were late endosomes or
lysosomes (Salonen et al. 2003). Thus it seems that endosomal targeting
is a joint action of nsPl and nsP3 domains in the nonstructural polypro-
tein. The polyprotein is attached to the membrane first by the nsPl
binding peptide, which adopts a-helical structure. Concomitantly the
nsPl domain undergoes a conformational change, which activates the
methyltransferase and guanylyltransferase. Palmitoylation of cysteine re-
sidues 418-420 thereafter anchors the protein irreversibly to the mem-
brane. We propose that the targeting of nsPl and polyproteins with this

Viral RNA Replication in Association with Cellular Membranes

149

* .»

'.i

* ■

Fig. 4. Immunolocalization of SFV nonstructural polyproteins expressed by the aid
of recombinant adenovirus vectors (A-C) and nsP3 during SFV infection (D) in
HeLa cells. Cleavage-deficient P12 CA (A) localizes to the plasma membrane and filo-
podia, whereas P12 CA 3 (B) displays vesicular staining. At the ultrastructural level
P12 CA 3 (C) localizes to the outer membrane of cytoplasmic vesicles (arrows), which
resemble the characteristic CPV structures carrying the viral replication complex in
SFV-infected cells (D). Bars 200 nm

domain to the plasma membrane may simply be dictated by the optimal
PS concentration in its cytoplasmic leaflet.

Early in alphavirus infection the minus-strand RNA synthesis is regu-
lated by the processing of the nonstructural polyprotein. The first cleav-
age releases nsP4 from PI 234 giving rise to the minus-strand polymer-
ase (Fig. 1A). The further processing of P123 is regulated by the slow

150

A. Salonen et al.

in cis cleavage of the nsPl/2 site, which is essential for the next cleavage
at the P2/3 site (Vasilieva et al. 2003). Thus the polyprotein has time to
fold properly and bind to membranes by the aid of the nsPl domain.
The proper folding of the complex enables protein-protein interactions,
which cannot be achieved when the components are expressed individu-
ally (Salonen et al. 2003). The polyprotein has a half-life of about 15 min
before it is processed into the final components. During this time a rep-
lication complex synthesizes possibly only one minus-strand RNA be-
fore it is transformed into a stable plus-strand polymerase, which oper-
ates as the unit of replication within the spherule (Kujala et al. 2001).

Alphavirus-Like Superfamily

Rubella virus belongs to the Togaviridae family together with al-
phaviruses, and rubella virus replication complexes resemble in many
ways those of SFV. Spherule-lined endo-lysosomal vacuoles are also
found in rubella virus-infected cells (Magliano et al. 1998). Rubella virus
replicase protein and newly synthesized RNA are located on the vac-
uoles, and specifically in spherule structures (Kujala et al. 1999). The
role of spherules as sites of RNA replication is supported by localization
of double-stranded RNA to them by antibodies against dsRNA (Lee et al.
1994).

Plant viruses belonging to the alphavirus-like superfamily replicate
on various intracellular membranes, for instance, brome mosaic virus
(BMV) and tobacco mosaic virus (TMV) on the ER, alfalfa mosaic virus
on the vacuolar (tonoplast) membrane, and turnip yellow mosaic virus
on the chloroplast envelope (Restrepo-Hartwig and Ahlquist 1996; Mas
and Beachy 1999; Prod'homme et al. 2001; van der Heijden et al. 2001).
For BMV (Fig. 5 A; Table 1), the targeting determinant of the replication
complex has been mapped to the la protein, and more precisely to its
N-terminal domain, part of which is distantly related to alphavirus
nsPl. la is peripherally but tightly bound to membranes and exposed to
the cytoplasm. Relatively large regions of la are needed for membrane
association and ER targeting, but the exact molecular basis for mem-
brane binding is not yet known (den Boon et al. 2001). Further compar-
ative studies are needed to determine whether replicase proteins in the
alphavirus-like superfamily share similar mechanisms of membrane as-
sociation and targeting, but in the case of several viruses the capping en-
zyme domain binds to membranes (Magden et al. 2001). Interestingly,

Viral RNA Replication in Association with Cellular Membranes

151

A. BMV 8.2 kb

Cap-

s' 5'

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Structural

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3Ainric

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Structural NS2 NS3 4A NS4B NS5A NS5B

D. EAV12.7 kb

5' ^ ^_^_^_^

Cap- nspl nsp2 | 3 | 4 | 5 1 1~7

p]//D-

poly(A)

Fig. 5A-D. Genome organization of model viruses described in the text, representing
the major groups of plus-strand RNA viruses. The different genomes are not in the
same scale, and the structural region of the EAV genome is not represented. Regions
attaching proteins to membranes are marked with star symbols (see also Table 1)

TMV replication in A. thaliana specifically and absolutely requires host
genes TOM1 or TOM3. They encode related multipass transmembrane
proteins, which seem to interact with the TMV replicase and are specu-
lated to participate in its membrane anchoring (Yamanaka et al. 2002).

In cells infected with these plant viruses structures closely resembling
the spherules, described above for alphaviruses, have been detected by
EM-techniques (Prod'homme et al. 2001; Schwartz et al. 2002 and refer-
ences therein). The spherules produced by BMV in yeast cells have re-
cently been characterized in exquisite detail (Schwartz et al. 2002). BMV
la protein alone, in the absence of other viral components, can induce
spherule formation. When viral RNA is coexpressed with la, it is appar-
ently protected inside the spherule in a membrane-associated, nuclease-
resistant state. When the viral polymerase protein 2a is coexpressed, it
associates with the spherules through interaction with la, and viral mi-
nus- and plus-sense RNA synthesis takes place in close association with

152

A. Salonen et al

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the spherules, possibly in their interior, from where plus-sense RNA is
released to the cytoplasm. Calculations based on immunolabeling sug-
gest that many (maximally a few hundred) la proteins may be present in
a spherule; leading to a hypothesis that la may form a shell-like struc-
ture coating the inside of the spherule (Schwartz et al. 2002). BMV repli-
cation in yeast requires a certain concentration of unsaturated fatty ac-
ids, as demonstrated through a mutation in the host fatty acid desatu-
rase gene and its complementation by addition of unsaturated fatty ac-
ids. Under restrictive conditions, la can still normally recruit viral RNA
and 2a to membranes, but minus-strand synthesis is strongly inhibited.
Unsaturated fatty acids, present in membrane lipids, generally increase
membrane fluidity and plasticity. Therefore, proper assembly or func-
tion of the BMV replication complex seems to require a relatively fluid
membrane (Lee et al. 2001).

Uniquely among positive-sense RNA viruses, a highly purified deter-
gent- solubilized replication complex of cucumber mosaic virus can cata-
lyze a complete cycle of minus-strand and plus-strand synthesis on an
exogenously provided specific template (Hayes and Buck 1990). Howev-
er, this preparation seems to be relatively unstable and it has not been
characterized further. In contrast, partially purified template-dependent
preparations, such as that isolated from TMV-infected cells (Osman and
Buck 1996), will be useful in further analyzing the role of membranes in
RNA replication.

Picornavirus-Like Superfamily

4.1

Poliovirus as a Model

Poliovirus is one of the best-characterized viruses. Its structure and rep-
lication have been described in recent reviews (Pfister et al. 1999;
Racaniello 2001; Semler and Wimmer 2002). Even though poliovirus
does not have an envelope, the synthesis of the structural and nonstruc-
tural proteins takes place in association with cytoplasmic membranes in
close vicinity to the RNA replication site (Caliguiri and Tamm 1970).
The entire positive-strand RNA genome is translated to a single polypro-
tein, which is nascently cleaved into three polyproteins, PI, P2, and P3.
PI is the precursor of virion structural proteins, whereas P2 and P3 re-
present nonstructural proteins participating in the replication of viral

Viral RNA Replication in Association with Cellular Membranes 155

RNA (Fig. 5B; Table 1). Protease 2A cleaves PI from the nascent polypro-
tein, while cleavages between nonstructural and structural proteins are
carried out by protease 3C pro (or rather 3CD pro ). Nonstructural protein
P2 yields protease 2A pro and 2BC, which in turn is cleaved to 2B and the
NTPase 2C. P3 yields 3AB and 3CDP ro , which are processed to 3A and 3B
(=VPg) and to 3C and 3D po1 , respectively (Fig. 5B). VPg, a terminal pro-
tein of 22 aa, is linked to the 5' end of the genome. The 5' nontranslated
region consists of a cloverleaf structure and an internal ribosome entry
site.

The incoming poliovirus genomes seem to migrate to specific perinu-
clear sites, where replication starts. RNA recombination, which occurs
during the synthesis of the complementary (minus-strand) RNA, takes
place in these perinuclear sites (Egger and Bienz 2002). Throughout in-
fection plus- and minus-strand RNAs are synthesized in the same repli-
cation complexes approximately in a ratio of 100 to 1 (Bolten et al.
1998). Poliovirus replication complexes consist of clusters of vesicles of
70-400 nm in diameter, which after isolation are associated as large
"rosettelike structures" of numerous vesicles interconnected with tubu-
lar extensions. The rosettes can dissociate reversibly into tubular vesi-
cles, which carry poliovirus nonstructural proteins on their surface and
synthesize poliovirus RNA in vitro (Semler and Wimmer 2002). Im-
munoisolated poliovirus-specific vesicles contain cellular markers for
the ER, lysosomes, and trans-Golgi network, suggesting complex bio-
genesis of the RNA replication complexes (Schlegel et al. 1996).

Involvement of the secretory route in the biogenesis of poliovirus rep-
lication complexes was suggested by the finding that brefeldin A, which
inhibits the transport of secretory proteins from the ER to the Golgi
complex, also inhibits poliovirus replication both in vivo and in vitro
(Racaniello 2001). The importance of ER as the primary source for po-
liovirus replication complexes was confirmed recently by experiments in
which COPII coat components were shown to colocalize with poliovirus
nonstructural proteins on budding vesicles upon their exit from ER.
Resident ER proteins were excluded from the released vesicles, which
were not destined to the Golgi complex, but accumulated in the cyto-
plasm (Rust et al. 2001). These results are in conformity with previous
findings, which showed that poliovirus infection inhibits the transport
of secretory and plasma membrane proteins (Doedens and Kirkegaard
1995). Thus it seems that in poliovirus-infected cells a continuous prolif-
eration and loss of ER membranes takes place. This process does not
supply the Golgi complex with its normal lipids. The sensitivity of polio-
virus replication to lipid synthesis inhibitors such as cerulenin could be

156

A. Salonen et al.

explained by this scenario (Racaniello 2001; Pfister et al. 1999). Overex-
pression of viral or cellular ER-associated proteins also inhibits poliovi-
rus replication, possibly by competing for the capacity of ER to generate
new membrane material (Egger et al. 2000).

Expression of P2 and P3 without structural proteins results in mem-
brane alterations similar to those seen in infected cells (Teterina et al.
2001). However, vesicles formed from nonreplicating poliovirus RNA
could not be recruited to support the replication of superinfecting polio-
virus RNA, suggesting that functional replication complexes are formed
only in cis by the direction of the incoming RNA. This must be first
translated to yield the replicase proteins for its own replication in situ
(Egger et al. 2000). Assuming that the newly synthesized plus-strands
create in turn new replication complexes by a similar in cis process, the
"rosette structures" might well consist of closely packed assemblies of a
parent replication complex and its numerous daughters, which are
loosely bound to each other (Semler and Wimmer 2002).

Numerous studies, in which poliovirus nonstructural proteins were
expressed individually or in combinations, in the absence of RNA syn-
thesis, have helped to understand the biochemical functions of non-
structural proteins and their role in membrane association during the
biogenesis of the replication complexes (Racaniello 2001; Semler and
Wimmer 2002). The 2B protein is targeted to ER membranes and to the
Golgi complex. It interferes with the secretory pathway in mammalian
and yeast cells (Barco and Carrasco 1995; Doedens and Kirkegaard 1995;
de Jong et al. 2003). It has been reported to disassemble the Golgi com-
plex (Sandoval and Carrasco 1997). 2B has a predicted cationic amphi-
pathic a-helix within the N-terminal 34-49 aa and a potential trans-
membrane domain (aa 61-81), which according to modeling form tetra-
meric aqueous pores, which could be responsible, for example, for the
observed hygromycin sensitivity and increased permeability of poliovi-
rus-infected cells. To cause these effects 2B has to be transported to the
plasma membrane, evidently on the cytoplasmic surface of the transport
elements (Agirre et al. 2002; de Jong et al. 2003).

When 2C is expressed alone in mammalian cells it is localized to the
ER, causing its expansion into tubular structures. As opposed to 2B, it
does not prevent the transport of VSV G-protein to the plasma mem-
brane (Suhy et al. 2000). The fragment responsible for the membrane
binding of 2C has been mapped to the N-terminal part of 2C within
aa 18-54 (Pfister et al. 1999). It has been predicted that this region has
an amphipathic a-helix, which starts either from residue 10 (Paul et al.
1994) or 21 (Echeverri and Dasgupta 1995). 2C has NTPase activity,

Viral RNA Replication in Association with Cellular Membranes 157

which can be inhibited by guanidine, the well-known specific inhibitor
of poliovirus replication (Pfister and Wimmer 1999). The ATPase activi-
ty of 2C is needed specifically in the initiation of minus-strand RNA syn-
thesis, the only step inhibited by guanidine (Barton and Flanegan 1997).
Because 2C binds specifically to the 3' end of the minus-strand RNA, it
may also have a function in the synthesis of the plus-strand RNAs, which
takes place even in the presence of guanidine, that is, without ATPase ac-
tivity. Anyhow, the tight membrane association of 2C and its intimate
participation in minus-strand RNA synthesis mean that this process
must also take place in association with membranes, although it has
been difficult to prove (Egger and Bienz 2002). 2BC, like 2B, is a mem-
brane protein, which interferes with the vesicular transport in both ani-
mal and yeast cells. Thus the 2B moiety in 2BC is responsible for the
transport inhibition (Doedens and Kirkegaard 1995). 2BC induces vesi-
cles similar to those seen in poliovirus-infected cells and causes perme-
ability increase of the plasma membrane, like 2B (Teterina et al. 1997).

3A expressed alone efficiently inhibits the vesicular transport of se-
cretory proteins from the ER to the Golgi. It remains associated with ER
membranes but can be mobilized into secretory vesicles, similar to those
in poliovirus-infected cells, by coexpression with 2BC (Dodd et al.
2001). In poliovirus-infected cells 3AB delivers the 22-aa-long VPg pep-
tide to the 5' end of both minus- and plus-strand RNA molecules. Only
membrane-associated 3AB can be cleaved by the viral proteases (3C pro
and 3CD pro ), and thus serve as the source of VPg (Pfister et al. 1999).
3AB associates tightly with cellular membranes, resembling the binding
of integral membrane proteins. The binding region has been mapped to
the C-terminal amino acids 59-80 of 3A, specifically to a hydrophobic
region consisting of aa 73-80. However, the exact binding mechanism is
not known (Towner et al. 1996). The 3B (=VPg) portion of 3AB has af-
finity to the catalytic subunit 3D po1 , and its precursor 3 CD, which in turn
recruits the template RNA into the membrane-associated replication
complex by interaction with 3C and 3D (Egger et al. 2000; Pfister et al.
1999).

In summary, the assembly of the poliovirus replication apparatus is a
complex process of specific membrane recognition, followed by protein-
protein and RNA-protein interactions. At the same time the vesicles de-
velop by a poorly understood autophagocytosis-like process to double-
membrane vesicles (DMVs) and large rosettes containing proteins from
ER, Golgi, and lysosomes (Schlegel et al. 1996; Suhy et al. 2000). Because
of the extreme proliferation of the ER, the secretory apparatus becomes
exhausted. The Golgi complex disappears, probably through retrograde

158

A. Salonen et al.

transport that is not compensated by normal lipid flow from the ER. De-
velopment of poliovirus-induced vesicles must be associated with multi-
ple fusion events directed either by viral or cellular proteins. Another
possibility would be that the membrane proteins from the Golgi com-
plex, and perhaps beyond it, would be enclosed to the poliovirus-specif-
ic vesicles through retrograde transport via ER. In any case, the result is
that the membranes of the secretory and endocytotic apparatuses be-
come mixed.

4.2

Other Members of the Picornavirus Superfamily

Many plant viruses in the picornavirus superfamily appear to replicate
in association with membranes derived from the ER. In comovirus- and
nepovirus-infected cells the ER is proliferated and vesiculized, but in
contrast to poliovirus-infected cells, the Golgi complex remains normal.
Replicase proteins and newly synthesized RNA are associated with the
ER-derived structures. Sensitivity to cerulenin, as an inhibitor of RNA
synthesis, seems to be a common property in the picornavirus super-
family (Carette et al. 2000; Ritzenthaler et al. 2002). Cowpea mosaic co-
movirus 32-kDa and 60-kDa replicase proteins are both targeted to sub-
regions of the ER when individually expressed, and they also cause mor-
phological alterations of the membrane system. The 32-kDa protein is a
hydrophobic component specific for comoviruses, whereas the 60-kDa
protein may contain membrane binding regions analogous to poliovirus
2C and 3A (Carette et al. 2002). For tobacco etch potyvirus, the 6-kDa
protein (analogous to poliovirus 3A) appears to be decisive in directing
the replicase to the ER. The 6-kDa protein associates tightly with ER
membranes by a single central hydrophobic domain (Schaad et al.
1997). The protein may be inserted to the membrane posttranslationally,
but its exact binding mechanism and topology are not known.

Although the polymerase of the insect nodaviruses appears to be dis-
tantly related to the picornavirus-like superfamily (Koonin and Dolja
1993), these viruses have capped mRNAs and the ultrastructure of the
replication complex resembles that of the alphaviruses. The outer mito-
chondrial membranes of flock house virus (FHV)-infected Drosophila
cells contain numerous spherulelike invaginations, connected to the cy-
toplasm by narrow necks. The number of spherules increases during in-
fection, leading finally to disruption of mitochondrial structure. The
single virus-encoded replicase component, 112-kDa protein A, localizes
to the outer mitochondrial membrane, which is also the site of viral

Viral RNA Replication in Association with Cellular Membranes 159

RNA synthesis (Miller et al. 2001). The N-terminal 46 aa of protein A
contain a mitochondrial targeting signal and a transmembrane helix,
such that the N-terminus is embedded in the mitochondrial matrix while
most of protein remains on the cytoplasmic side (Miller and Ahlquist
2002). This transmembrane topology distinguishes nodavirus replicase
from the replicase proteins of alphaviruses.

An interesting result has been obtained with the crude membrane-
bound replication complex isolated from FHV- infected cells. When treat-
ed with micrococcal nuclease and supplied with an exogenous template,
the FHV replicase synthesizes a complementary minus-strand resulting
in a dsRNA product. However, when glycerophospholipids are added to
the mixture, relatively large quantities of plus-strand RNAs are also pro-
duced, that is, a complete RNA replication cycle takes place. Several
phospholipid species can stimulate this reaction, for instance, phospha-
tidylcholine bearing acyl chains of 14-18 carbons. It has been speculated
that glycerophospholipid might directly interact with a component of
the crude membrane preparation, perhaps activating an enzymatic func-
tion, or alternatively, that the lipid might facilitate a membrane modifi-
cation or assembly process required specifically for plus-strand synthe-
sis (Wu et al. 1992). Because of these advances and the simplicity of the
nodavirus replicase, this virus group is promising for further analysis of
membrane-associated replication.

Flavivirus-Like Superfamily

Of the members of the Flaviviridae, hepatitis C virus (HCV) and Kunjin
virus are the best studied in the context of membrane-associated replica-
tion. Here they will be discussed only briefly, because these aspects of
HCV and Kunjin virus have been reviewed recently (Dubuisson et al.
2002; Westaway et al. 2002).

Analogous to picornaviruses, the whole positive-strand RNA genome
of flaviviruses is translated to a large polyprotein. The structural pro-
teins consist of a capsid protein and envelope glycoproteins followed by
nonstructural proteins (Fig. 5C; Table 1). Because the envelope proteins
are translocated to and glycosylated in the ER, it would be expected that
the nonstructural proteins would associate directly to the ER membrane.
Nevertheless, in heterologous expression systems HCV nonstructural
proteins NS2, NS4A, NS4B, NS5A, and NS5B each alone bind to the ER,
whereas the soluble protease/helicase (NS3) associates with the mem-

160

A. Salonen et al.

brane by interaction with NS4A, a cofactor for the protease domain of
NS3 (Dubuisson et al. 2002; Wolk et al. 2000).

NS2 is a polytopic integral membrane protein introduced to the ER
membrane by internal signal sequences. It is a protease responsible for
the cleavage between NS2 and NS3, but it is not essential for RNA repli-
cation (Yamaga et al. 2002). NS4B is also a polytopic membrane protein
cotranslationally inserted into the ER membrane with its own internal
signal sequences (Hugle et al. 2001). Although the exact function of
NS4B is not known, its interaction with NS3 and NS5B modulates the
RNA polymerase activity (Piccininni et al. 2002). NS5A phosphoprotein
is tightly associated with membranes through an N-terminal amphipath-
ic helix of about 30 residues. When these residues are joined to GFP, the
fusion protein is associated with ER membranes, suggesting that the N-
terminus of NS5A has also an address for the ER, in addition to mem-
brane binding (Brass et al. 2002; Dubuisson et al. 2002). The monotopic
binding of NS5A to membranes resembles the situation in alphaviruses,
except that the binding peptide of NS5A is "tip -anchored," rather than
residing in the middle of the protein like in nsPl (Ahola et al. 1999).
NS5B, the catalytic subunit of the HCV RNA polymerase, has a C-termi-
nal membrane insertion sequence of 21 aa, which is targeted to the ER
membrane posttranslationally, like typical tail-anchored membrane pro-
teins (Ivashkina et al. 2002).

Expression of the entire HCV polyprotein induced a special ER-de-
rived membranous web, where a cluster of tiny vesicles was embedded
in a membranous matrix, often accompanied by tightly associated vesi-
cles surrounding the web. According to immuno-EM analysis, all HCV
proteins were associated with the web, but not with the vesicles. When
the ns proteins were expressed individually or in combinations, NS4B
alone induced the web, whereas NS3-NS4A complex induced a multitude
of single vesicles having no direct analog in polyprotein-expressing cells
(Egger et al. 2002). NS5A and NS5B did not modify the ER. In cells ex-
pressing functional HCV subgenomic replicons all ns proteins associat-
ed with ER membranes according to light and electron microscopic
analysis (Mottola et al. 2002).

Even though the genome organization of HCV and flaviviruses is sim-
ilar, there are some differences as well. Flaviviruses have a nonstructural
glycoprotein NS1 (46 kDa), which is translocated into the lumen of ER
and transported through the secretory route to the exterior of the cell.
NS1 also plays an essential role in RNA replication, evidently by recog-
nizing portions of the other replicase proteins penetrating the ER
membrane (Westaway et al. 2002). Another difference is that there are

Viral RNA Replication in Association with Cellular Membranes 161

two small membrane-associated proteins, NS2A (25 kDa) and NS2B
(14 kDa), preceding the soluble NS3 protein (60 kDa), which has prote-
ase-helicase-RNA triphosphatase activities. NS2B acts as a cofactor for
the NS3 protease. NS4A (16 kDa) and NS4B (27 kDa) are poorly con-
served membrane proteins. Finally, NS5 (104 kDa) protein is the catalyt-
ic subunit of the RNA polymerase complex. It is a soluble protein, unlike
its homologous counterpart NS5B of HCV.

Extensive immunofluorescence microscopy studies of Kunjin virus-
infected cells have established that NS1, NS2A, NS3, NS4A, and NS5 are
regularly associated with dsRNA, which has served as marker for gen-
uine replication complexes. These markers also colocalize with cellular
markers of trans-Golgi membranes (Mackenzie et al. 1999), even in cells
that do not express the viral glycoproteins (Mackenzie et al. 2001).

Electron microscopy of Kunjin virus-infected cells has revealed dra-
matic changes in the organization of the ER membrane. Proliferation of
the ER leads to convoluted membranes (CM) and paracrystalline struc-
tures (PC) and to vesicle packets of smooth membranes ( VP) ( Westaway
et al. 1997, 2002). The majority of NS proteins, dsRNA, as well as nascent
labeled viral RNA have been immunolocalized to VPs, which are derived
from trans-Golgi membranes late in infection. VPs are "vesicle sacs"
consisting of a cluster of individual vesicles (diameter about 50-100 nm)
surrounded by a membrane. Interestingly, VPs were not detected on ex-
pression of Kunjin replicons, whereas CMs and PCs were formed. In
these cells the dsRNA was scattered throughout the cytoplasm in small
isolated foci, suggesting that all membrane structures induced by the
replicase proteins are not necessarily sites of RNA replication (Mackenzie
et al. 2001; Westaway et al. 1999). Comparison between replicon cell lines
producing RNA and NS proteins with different efficiencies suggests that
the induction of virus -specific membranes is dose dependent and re-
quires a certain level or concentration of viral products to manifest
(Mackenzie et al. 2001).

Tombusviruses, plant viruses classified in the same supergroup with
flaviviruses, replicate on peroxisomal or chloroplast membranes, or on
the mitochondrial outer membrane depending on the virus species.
Tombusvirus infection induces multivesicular bodies, where the limiting
membrane of the organelle is transformed into numerous spherules
(Rochon 1999). A putative polymerase of carnation Italian ringspot virus
is a 92-kDa protein translated by read-through of an amber termination
codon at the end of a 36-kDa protein. Both the 36-kDa and 92-kDa
proteins are targeted to the mitochondrial membranes and anchored
there via two hydrophobic domains located close to the N-terminus

162

A. Salonen et al.

(Weber-Lotfi et al. 2002). However, the expression of the 36-kDa protein
alone was not sufficient to induce the vesiculation of mitochondria and
hence the formation of spherules (Rubino et al. 2000).

Nidoviruses

Coronaviruses and arteriviruses, which are grouped in the order Nidovi-
rales, express their replicase genes from two large open reading frames
through complex proteolytic processing, leading to 12 or more end
products, depending on the virus (Snijder and Meulenberg 2001; Lai
and Holmes 2001). The replicase proteins of equine arteritis virus (EAV)
(Fig. 5D; Table 1), as well as newly synthesized RNA, accumulate in peri-
nuclear granules and vesicles, which are of ER origin (van der Meer et
al. 1998). An electron microscopic study of EAV-infected cells revealed
DMVs of approximately 80-nm diameter, carrying the replication com-
plex (Pedersen et al. 1999). Usually the inner and outer membranes of
the DMVs were tightly apposed but clearly separate. The mechanism for
DMV formation appears to be a protrusion of paired ER-membranes,
because DMVs having the outer membrane continuous with ER could
be seen. These profiles sometimes contained a neck between the paired
ER-membrane and a forming DMV, which had not yet pinched off. The
formation of DMVs is not dependent on RNA synthesis, because DMVs
strikingly resembling those seen in EAV-infected cells can be induced by
heterologous expression of the nsp2-nsp3 region of the polyprotein
(Snijder et al. 2001). On individual expression of these proteins, DMVs
were not observed. The large nsp2 has a long central hydrophobic se-
quence, which may represent its membrane anchor, whereas nsp3 and
nsp5 have several hydrophobic sequences, suggesting that they are poly-
topic membrane proteins. They all, and their precursors, also behave
biochemically as integral membrane proteins (van der Meer et al. 1998).
DMVs are also the sites of coronavirus RNA replication (Gosert et al.
2002). Coronavirus-induced DMVs are larger than those induced by EAV,
over 200 nm in diameter, and they are surrounded by tightly apposed
membranes that have fused into a lipid trilayer. Viral RNA and replicase
proteins are found on the surface of DMVs, where RNA synthesis also
takes place. The coronavirus replicase proteins are membrane associated
and include the integrally bound components p210 and p44, the corona-
virus counterparts of EAV nsp2 and nsp3 (Gosert et al. 2002). At the
moment, the origin of the coronavirus replication complexes (DMVs) is

Viral RNA Replication in Association with Cellular Membranes 163

not clear; involvement of membranes from secretory and endosomal
compartments has been suggested. It should be noted that the majority
of the viral replicase proteins may be located elsewhere than in the active
replication complexes, as in SFV-infected cells (Kujala et al. 2001).

Concluding Remarks
7.1

Targeting of the Replicase Complex

Many viruses replicate on the cytoplasmic side of the ER membrane. For
instance, picornavirus nonstructural proteins are targeted to the ER
through 2B, 2C, 3A, and their precursors, bromovirus replication com-
plex by the la protein, and arterivirus replicase by the nsp2-nsp3 com-
plex (Table 1). However, the targeting mechanisms remain to be solved,
as no specific receptors for viral components on the ER have been iden-
tified. Because all HCV and flavivirus proteins are translated from the
same polyprotein as their envelope glycoproteins, it might be expected
that their ns proteins also remain at the ER membrane. However, their
location is guaranteed by their own independent affinity for ER mem-
branes. In the case of Kunjin virus the NS1 glycoprotein, which is
translocated to the cisternal side of the ER, may be responsible for the
transport of the replication complex to the trans-Golgi region by trans-
membrane contact.

For some viral replicase proteins, classic targeting sequences direct-
ing them to a specific compartment have been identified. FHV virus
protein A is directed to the mitochondrial outer membrane by a specific
targeting sequence. A similar mechanism seems to operate in the target-
ing of the tombusvirus replication complex to either mitochondria or
chloroplasts, depending on the virus species. The RNA replication of al-
phaviruses on the membranes of the endosomal apparatus (plasma
membrane, endosomes, and lysosomes) might be explained by direct in-
teraction of the amphipathic a-helix of nsPl with these PS-rich mem-
branes. It is an interesting question whether specific lipid components,
or the lipid composition of the target membranes, might attract repli-
case components of other viruses as well.

The genomes of animal viruses discussed in this review are expressed
as polyproteins. Picornaviruses and flaviviruses express both structural
and nonstructural proteins in the same polyprotein, whereas togaviruses

164

A. Salonen et al.

and nidoviruses express their nonstructural proteins as a separate
polyprotein. The polyprotein strategy evidently guarantees the proper
targeting and assembly of the membrane-associated RNA replication
complex. For instance, in SFV-infected cells the delayed proteolysis of
the ns polyprotein enables the folding and assembly of "soluble" compo-
nents (nsP2-nsP4) with the nsPl membrane anchor. In the case of picor-
naviruses and flaviviruses several membrane anchors are present.

7.2

Mechanisms of Membrane Binding

The modes of membrane attachment are also variable. Monotopic bind-
ing by amphipathic a-helix has been suggested for several replicase pro-
teins, which lack continuous hydrophobic anchor sequences (Table 1).
Except for SFV nsPl (Lampio et al. 2000) and HCV 5 A (Brass et al.
2002), these conclusions have been based only on sequence-based pre-
dictions and mutagenesis studies. The amphipathic a-helix strategy has
been proposed for components of picornavirus and for several plant vi-
rus replication complexes. Monotopic hydrophobic anchors have been
proposed for poliovirus 3A and related plant virus proteins, and poly-
topic membrane anchors have been demonstrated for HCV proteins NS2
and NS4B. Recent results have shown that the catalytic subunit of HCV
RNA polymerase is a typical tail-anchored ER protein, whereas the poly-
merases of other viruses are soluble proteins (Table 1).

7.3

Membrane Modification by Replicase Proteins

Alphaviruses and rubella virus give rise to specific cytoplasmic vesicles
with regular membrane invaginations, spherules (Fig. 6 A and B), that

►

Fig. 6A-E. Ultrastructure of membranes involved in virus replication. A Typical cyto-
pathic vesicles (CPVs) in SFV-infected BHK cell (4 h p.i.) with characteristic spher-
ules inside. B A single spherule showing a neck opening to the cytoplasm with filled
electron-dense material (courtesy of Dr. Ari Helenius and Dr. Jurgen Kartenbeck).
C Poliovirus type 1 -infected COS-1 cell (4 h p.i.) showing typical cytoplasmic vesi-
cles ranging from 70 to 400 nm (courtesy of Dr. Karla Kirkegaard and Dr. Thomas
Giddings). D EAV-infected BHK cell (4 h p.i) showing double membrane vesicles
(DMVs) in close vicinity to RER (courtesy of Dr. Eric Snijder and Dr. Ketil Pedersen).
E Membraneous web in UHCV-57.3 cell expressing the entire HCV open reading
frame, 48 h after tetracycline removal (courtesy of Dr. Kurt Bienz). Bars 200 nm (A),
60 nm (B), 100 nm (D), 500 nm (E)

Viral RNA Replication in Association with Cellular Membranes

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seem to be the actual units of RNA replication (Kujala et al. 2001). So
far, it is not known how these structures arise. Similar spherules have
been described in BMV-infected plant and in yeast cells expressing non-
structural proteins of the virus. It has been proposed that the internal
surface of the spherules is covered by the replicase protein la of BMV
(Schwartz et al. 2002). As the spherules seem to be a general feature for
the members of the alphavirus superfamily, it will be interesting to see
whether the suggested protein coating of the inner surface of spherules
is a general mechanism within the whole superfamily. Involvement of
host cell proteins cannot be excluded, as membrane bending and vesicu-
lation in cells is a complex process requiring several factors (Hurley and
Wendland 2002).

Membrane vesicles and multivesicular bodies induced by poliovirus
(Fig. 6C) and other picornaviruses contain proteins from the Golgi com-
plex, endosomes, and lysosomes, suggesting multiple fusion events dur-
ing their development. Isolated membrane structures, "rosettes," consist
of clusters of vesicles, which can be separated from each other at low
ionic strength and low temperature. The individual vesicles represent
units of replication (Egger et al. 1996). They have a tubular extension,
which resembles the neck of a spherule through which the nascent RNA
is proposed to be "secreted" from the site of synthesis (Froshauer et al.
1988). The DMVs (Fig. 6D) seem to the sites of arterivirus and coronavi-
rus RNA synthesis as well. Evidently the vesicles wrap the template
RNA, which in the case of poliovirus, alphaviruses, and flaviviruses is
probably double-stranded, shielding it from host nucleases. During vi-
rus infection, translation and assembly of virus particles take place in
close association with the replication complexes, utilizing nascent RNAs
immediately after their synthesis.

A common feature for the viruses discussed in this review is that the
nonstructural proteins, in the absence of RNA template, seem to be suf-
ficient to induce the membrane modifications seen in the infected cells.
Such is also the case for the membranous web seen during HCV
polyprotein expression (Fig. 6E). Future studies at the molecular level
should reveal how the different viruses and proteins can cause these fun-
damental structural changes in the membranes. A suitable lipid compo-
sition of the membrane may also be required for these dynamic mem-
brane assembly and modification processes (Lee et al. 2001).

Viral RNA Replication in Association with Cellular Membranes 167

7.4

Functional Implications of Membrane Attachment

As the polymerase complex itself is firmly attached to the membrane,
the template apparently has to move through the complex, which often
also contains helicase, capping enzyme, and other subunits. In most in-
stances, this would also mean that the same template would be repeated-
ly utilized by circling through the same replication complex. The dimen-
sions of the membrane vesicles seen in EM images are such that the
RNA would be relatively tightly packed within them, analogous to
dsRNA virus cores. For picornavirus-like viruses there are special chal-
lenges, as for each round of RNA synthesis a protein component is con-
sumed as a primer. It should also be emphasized that membrane lipids
provide active components for the replication complex. They may di-
rectly bind to replicase proteins, thereby changing their conformation
and activating them (Ahola et al. 1999).

Acknowledgements We thank Drs. Eija Jokitalo for the EM figures and Ilkka Kilpelainen
for help in presenting the peptide structure and Marja Makarow for critical reading
of the manuscript. The work has been supported by the Academy of Finland (grants
8397 and 201687), Biocentrum Helsinki, and Helsinki University Research Funds.
Drs. Kurt Bienz (University of Basel), Ari Helenius, (ETH, Zurich), Jurgen Karten-
beck (DKFZ, Heidelberg), Karla Kirkegaard (Stanford University), Thomas Giddings
(University of Colorado), Eric Snijder (University of Leiden), and Ketil Pedersen
(University of Oslo) are gratefully acknowledged for the electron micrographs.

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CTMI (2004) 285:175-198
© Springer- Verlag 2004

Synthesis and Quality Control
of Viral Membrane Proteins

C. Maggioni • I. Braakman ([*])

University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands
I.Braakman@chem.uu.nl

1 Introduction 176

2 Protein Biosynthesis 176

2.1 Entering the Endoplasmic Reticulum: Targeting and Translocation ... 176

2.2 Protein Folding in the ER 178

2.2.1 Chaperones and Folding Enzymes 178

2.2.2 Glycosylation and the CNX/CRT Cycle 182

2.2.3 Disulfide Bond Formation 184

3 Quality Control 185

3.1 Retention in the ER 185

3.2 Degradation and the Unfolded Protein Response 186

4 How Viruses Evolve to Fool the Host 188

5 Conclusions 191

References

191

Abstract Viruses use the host cellular machinery to translate viral proteins. Similar
to cellular proteins directed to the secretory pathway, viral (glyco)proteins are syn-
thesized on polyribosomes and targeted to the endoplasmic reticulum (ER). For
viruses that encode polyproteins, folding of the individual proteins of the precursor
often is coordinated. Translocation and the start of folding coincide and are assisted
by cellular folding factors present in the lumen of the ER. The protein concentration
a newborn protein finds in this compartment is enormous (hundreds of mg/ml) and
the action of molecular chaperones is essential to prevent aggregation. Viral envelope
proteins also undergo the cellular quality control mechanisms, which ensure, with
variable stringency, that only proteins with the correct structure will proceed
through the secretory pathway. Proteins that are misfolded, or not yet folded, are re-
tained in the ER until they reach the native conformation or until their retrotr allo-
cation into the cytosol for degradation. Peculiar characteristic of viruses is their abil-
ity to interfere with the cellular machinery to ensure virus production and, more-
over, to pass through the body unobserved by the host immune system. This section
describes some mechanisms of genetic variation and viral immune evasion that in-
volve the secretory pathway.

176

C. Maggioni • I. Braakman

Introduction

The membranes of enveloped virus particles contain one or more types
of virally encoded integral membrane protein. The most abundant of
these proteins contain a single transmembrane domain, a large ectodo-
main, that is localized at the outside of the viral envelope and is frequent-
ly glycosylated, and a small cytoplasmic domain. Almost all viral mem-
brane proteins are oligomeric, either homo-oligomeric or hetero-
oligomeric. Each subunit consists of one or two polypeptide chains that
can be held together by noncovalent interactions or by covalent disulfide
bridges. In the case of two polypeptide chains, they are often derived
from a single-chain precursor that is proteolytically cleaved during trans-
port of the protein to the plasma membrane. The cleavage is accompanied
by conformational changes that prime the biological activity. For most of
the viral proteins the cleavage is performed in trans by cellular or viral
proteases. Viral envelope proteins have different functions: They mediate
binding to the receptor(s) on the host cell's plasma membrane, mem-
brane fusion, and penetration. For some viruses all these functions are
combined in a single glycoprotein. In addition, some small viral envelope
proteins have been shown to possess ion channel activity (reviewed by
Fischer and Sansom 2002). The M2 of Influenza A for example, possesses
proton conductance activity and is responsible for a pH change in the in-
terior of the virion that is necessary for disassembly of the virus particle.
This class of viral membrane proteins contains one or more transmem-
brane domains and either lacks or has a lower number of glycans and di-
sulfide bonds. To be functional, as is true for all proteins, viral envelope
glycoproteins must reach the correct three-dimensional structure, a pro-
cess that is referred to as protein folding. To translate and fold viral enve-
lope proteins, viruses use the host cellular machinery and they are sub-
jected to the same quality control systems as endogenous proteins.

Protein Biosynthesis
2.1

Entering the Endoplasmic Reticulum: Targeting and Translocation

The biogenesis of most secretory and membrane proteins involves tar-
geting of the nascent protein to the endoplasmic reticulum (ER), trans-
location across or integration into the ER membrane, and maturation

Synthesis and Quality Control of Viral Membrane Proteins 177

into a functional product. Leader peptides of nascent chains are recog-
nized in the cytosol by signal recognition particle (SRP) as soon as they
emerge from the ribosome. The SRP-nascent chain-ribosome complex
interacts with the SRP receptor, which targets it to the ER. Once target-
ed, the nascent chain is transferred to the translocation channel in a
GTP-dependent step. The ribosome-nascent chain complex is tightly
bound to the translocon without the ER luminal and cytosolic contents
coming into communication. The translocation channel is the het-
erotrimeric Sec61p complex, composed of a-, /?-, and y-sub units (re-
viewed by Matlack et al. 1998). The channel is not a passive hole but is a
dynamic structure that accomplishes different functions including tar-
geting, cotranslational translocation, and cotranslational integration
(Johnson and van Waes 1999).

Other proteins that are part of the translocation machinery are the
translocon-associated protein (TRAP) complex with unknown function
(Hartmann et al. 1993; Wang and Dobberstein 1999), the translocating
chain associating membrane protein (TRAM), the small ribosome-asso-
ciated membrane protein 4 (RAMP4) (Gorlich and Rapoport 1993), the
signal peptidase (SP), and oligosaccharyl transferase (OST). TRAM is
necessary for translocation of most substrates and regulates which do-
mains of the nascent chain are "accessible" from the cytosol during a
translocational pause (Hegde et al. 1998). Signal peptidase is necessary
for cleavage of leader peptides and OST for transfer of oligosaccharide
chains to nascent proteins, two processes that as a rule occur co-
transl(oc)ationally. Exceptions to this rule have been found for some vi-
ral proteins. The leader peptide of the HIV-1 Envelope protein (Berman
et al. 1988; Li et al. 1994; Land et al. 2003) and the signal peptide of a
portion of the newly synthesized HCMV US 11 (Rehm et al. 2001) are re-
moved only after synthesis of these proteins is completed. Glycosylation
of the hepatitis C virus (HCV) envelope protein El can occur also post-
translationally, albeit in a mannosylphosphoryldolichol-deficient CHO
mutant cell (Duvet et al. 2002). Because molecular details of biosynthetic
processes are more frequently studied in viral proteins than in mam-
malian proteins, we can only speculate on the specificity of these excep-
tions for viral proteins, and on their abundance in both the viral and the
mammalian world.

Not only the translocon and the associated proteins but also ER lumi-
nal proteins have been shown to be necessary for proper translocation.
For example BiP, the Hsp70 homolog in the ER, was found to be respon-
sible for sealing the luminal end of not only the ribosome-free but also
the ribosome-docked translocon (Haigh and Johnson 2002).

178

C. Maggioni • I. Braakman

2.2

Protein Folding in the ER

The ER is the entry point for newly synthesized proteins that are direct-
ed into the secretory pathway. Folding can occur spontaneously because
all information needed to reach the proper three-dimensional structure
is present in the primary sequence (Anfinsen 1973). Still, within the cell
the process needs assistance, which is provided by helper proteins
known as molecular chaperones and folding enzymes. The ER is special-
ized for protein folding, providing an optimized environment: of oxidiz-
ing conditions and a high concentration of chaperones and folding en-
zymes. Folding begins cotranslationally and continues for minutes to
hours after termination of polypeptide synthesis.

Some viral glycoproteins need not only cotranslational folding but
also cotranslational assembly to reach their proper, native structure. For
example, correct folding of the envelope glycoprotein El of HCV re-
quires the presence of E2 (Michalak et al. 1997) through an interaction
between the transmembrane domains of the two proteins (Cocquerel et
al. 2001; Patel et al. 2001). In addition, another flanking protein of El,
the core protein, seems to play a role for correct folding of El (Merola et
al. 2001). The folding process of p62 and El, the envelope glycoproteins
of Semliki Forest virus, is coordinated as well. The p62 protein can effi-
ciently fold without El, but El is found in aggregates in the absence of
p62 (Andersson et al. 1997). Sindbis, another alphavirus, has an enve-
lope composed of 80 trimers of E1-E2 dimers. The precursor of E2, pE2,
dimerizes with El but, in this case, p62 interaction is needed for assem-
bly and exit from the ER rather than for proper folding of El (Carleton
et al. 1997). A recent study on the flavivirus tick-borne encephalitis
(TBE) virus (Lorenz et al. 2002) clearly shows perhaps the first example
of a mutual need for proper folding of the two envelope glycoproteins E
and the precursor of M (prM), suggesting a chaperone-like function for
prM. At the same time, E is also necessary for rapid signal sequence
cleavage of prM and the two proteins can be each other's helpers even
when expressed in trans, from different constructs.

2.2.1

Chaperones and Folding Enzymes

The main challenge a newly synthesized protein encounters within a cell
is the intracellular environment, in particular the extremely high protein
concentration of more than 200 mg/ml (Ellis 2001). A nascent chain is

Synthesis and Quality Control of Viral Membrane Proteins 179

prone to aggregation and misfolding because of this high concentration
(molecular crowding) and because of the close proximity of other na-
scent polypeptides emerging from a polyribosome. Considering that ri-
bosomes on the same mRNA were found at a distance of only 80 nucleo-
tides (Hesketh and Pryme 1991), or even 27 nucleotides during ribo-
some pausing (Wolin and Walter 1988), nascent chains in the lumen of
the ER should emerge at a distance of about 15-40 nm, corresponding
to a chain length of only a hundred amino acid residues or less. In the
living cell, nonproductive protein folding generally is prevented by the
action of molecular chaperones. Two types of chaperones can be recog-
nized: the general kind, with a highly promiscuous interaction pattern,
and the private kind, which caters for one particular protein (family)
only.

In the ER, these general chaperones, present also at high concentra-
tion (Fig. 1A), associate with the growing nascent chain during translo-
cation and continue to assist folding until a protein has acquired its na-
tive structure. Molecular chaperones act by facilitating rate-limiting
steps, by stabilizing unfolded proteins, and by preventing undesired in-
ter- and intrachain interactions that could lead to aggregation. They
may recognize hydrophobic surface patches, mobile loops, and lack of
compactness, or they may recognize a specific amino acid sequence in
the case of the private chaperones. These features are exposed transient-
ly during folding, resulting in only transient associations with ER fold-
ing factors if the newly synthesized protein folds correctly.

The general chaperones identified so far in the ER are among the
most abundant proteins in a cell, and even more so in the ER. Well-
known and well-studied are the ER Hsp70 homolog BiP, the Hsp90 ho-
molog Grp94, and the lectin chaperones calnexin (CNX) and calreticulin
(CRT). Long-known folding enzymes include protein disulfide isome-
rase (PDI) and two other oxidoreductases, ERp72 and ERp57. More re-
cently, ERp44 was identified in mammalian ER (Anelli et al. 2002), and
the Saccharomyces cerevisiae genome revealed the existence of a total of
five PDI family members in the yeast ER (Norgaard et al. 2001). Pep-
tidyl-prolyl cis-trans isomerases (PPIases) constitute another class of en-
zymes present in the ER. They catalyze the isomerisation of peptide
bonds between any amino acid and a proline in a polypeptide chain;
their role in folding was mainly demonstrated in vitro. A very high num-
ber of PPIases were discovered in mammals, grouped in three families
(cyclophilins, FK506 binding proteins, and parvulins). Their functions
reach beyond the assistance of newly synthesized proteins, being at the
intersection between protein folding, signal transduction, trafficking, as-

180

C. Maggioni • I. Braakman

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Synthesis and Quality Control of Viral Membrane Proteins 181

sembly, and cell cycle regulation (Gothel and Marahiel 1999). Mechanis-
tic details of some of the ER folding factors are described below.

The question of why particular newly synthesized proteins have a
preference for particular chaperones and folding enzymes has not been
answered yet. Helenius and colleagues postulated that a hierarchy exists
that depends on the position of the first glycan (Molinari and Helenius
2000). Important is the concept of redundancy. It exists everywhere in
biology and is prominent among chaperones and folding enzymes. If the
favored chaperone or folding enzyme is not available (because of a de-
fect or because of competition) another ER folding factor takes over.
This second folding factor may act through a different mechanism, but
the end result, a high yield of properly folded protein, may be the same.
A consequence of redundancy is the lower chance for misfolding, and at
the same time an increased likelihood of competition. The ER protein
folding capacity therefore is likely to depend on the relative concentra-
tion of the various ER folding factors, as well as the nature of the pro-
teins synthesized by that cell.

Viruses have evolved to exploit the cell's machineries, including those
specialized in protein folding and quality control. Some viruses may
have strategies to stop host protein synthesis altogether, but any virus
will cause a shift in the balance in the ER. Viral proteins as a rule are
much more abundant than endogenous newly synthesized proteins, such
that they compete out other substrates for the ER folding factors. This is
clearly illustrated when comparing the number of proteins coimmuno-
precipitated with CNX from a noninfected cell lysate with the almost ex-
clusive coimmunoprecipitation of X31 influenza virus hemagglutinin
(HA) from an influenza-infected cell lysate (Peterson et al. 1995), even
though host protein synthesis is barely inhibited by this influenza
strain.

The private chaperones are protein-specific factors that have evolved
to assist the folding of protein (families) with atypical physical struc-
tures, or of proteins that exist in unusual situations or conditions. Colla-

Fig. 1. Inside the crowded ER. A Model for ER crowding: in the lumen of the ER,
newly synthesized proteins are received into a very crowded environment. Proteins
are depicted according to their relative size and shape. B Venn diagram showing the
overlapping functions of some folding factors. The inset shows one of the known
processes in protein folding: the formation of disulfide bonds in a nascent protein.
The final electron acceptors (gray box) have been shown for yeast but are still un-
clear for mammalian cells

182

C. Maggioni • I. Braakman

gen, for example, needs HSP47 to prevent premature fiber formation
(Nagata 1996; Tasab et al. 2000), and RAP is considered to be specific for
the LDL receptor family (Bu et al. 1995). Another example is the ER-res-
ident molecule tapasin (Tpn) that is uniquely dedicated to tethering
MHC class I molecules jointly with the chaperone calreticulin and
the oxidoreductase ERp57 to MHC-encoded peptide transporter TAP
(Momburg and Tan 2002).

Whereas viral proteins are found abundantly associated with general
folding factors, they are of course less likely to use such private chaper-
ones, except when they would be virally encoded. On the other hand, we
cannot exclude that viral proteins coevolving with cells acquire(d) or ex-
ploit private chaperones. As yet, the number of proteins (both viral and
mammalian) for which biosynthesis is extensively studied is too low to
allow general conclusions.

2.2.2

Glycosylation and the CNX/CRT Cycle

Glycosylation has been shown to be important for protein folding, for
protein stability, for immune evasion, and for receptor usage. Most viral
envelope proteins are glycoproteins, and they bind during folding to the
lectin chaperones CNX and CRT (Parodi 2000). The process of N-linked
glycosylation of secretory proteins is characterized by enzymatic reac-
tions occurring on both sides of the ER membrane. Monosaccharides
are added to the lipid intermediate dolichol pyrophosphate to make
Man5GlcNac2-PP-Dol. Further elongation of the glycan chain depends
on specific translocators called flippases (Helenius et al. 2002). Once
"flipped" over to the ER luminal side, the precursor is elongated to
Glc3Man9GlcNac2-PP-Dol, for which the OST complex has optimal af-
finity. The enzyme transfers the oligosaccharide 14-mer en bloc to as-
paragine residues on the nascent chain within the consensus sequence
Asn-X-Ser or Asn-X-Thr, with X being any amino acid except proline or
aspartic acid.

General dogma says that glycans are crucial for proper glycoprotein
folding. The reasons are understood to a large extent. Addition of a gly-
can adds a very large, strongly hydrophilic moiety to an otherwise per-
haps more hydrophobic stretch of polypeptide. This will force this re-
gion of the protein away from the hydrophobic core of the protein. An-
other, direct, effect of glycosylation was found in peptides. A glycosylat-
ed peptide was shown to adopt a different conformation than the peptide
without glycan (Imperiali and Rickert 1995), indicating a direct change

Synthesis and Quality Control of Viral Membrane Proteins 183

of protein structure. Studies with the glucosidase inhibitor n-butyl de-
oxynojirimycin (nBuDNJ) showed that the assembly of viral envelopes
of viruses, such as for example HIV, was not affected. However, the virus
could not enter the host cell because the rearrangement of the V1/V2
loops necessary to release gpl20 from gpl60 could not take place (Fi-
scher et al. 1996) with gp41 unable to mediate fusion. This demonstrates
the importance of correct glycosylation for viral proteins.

A more indirect role for glycans is their recognition by the lectin
chaperones CNX and CRT. These proteins bind the carbohydrate chain
of newly synthesized glycoproteins only when in the monoglucosylated
form, reached after trimming of the N-linked glycan by action of ER
glucosidases I and II. A prolonged interaction with CNX and CRT is es-
tablished via reversible glucosylation of the N-linked glycan after gluco-
sidase II has removed the important glucose. In this way, CNX and CRT
bind and release substrate in cycles, giving a prolonged chance to the
substrate to fold correctly. The enzyme responsible for reglucosylation
is the UDP-glucose:glycoprotein glucosyltransferase (GT), which be-
haves as a sensor for glycoprotein conformation (Parodi 2000; Ellgaard
and Helenius 2001). The interaction with CNX and CRT exposes the
folding protein to the associated cochaperone ERp57, a thiol oxidore-
ductase of the PDI family. During folding of viral glycoproteins, ERp57
has been shown to form transient intermolecular disulfide bonds with
glycoprotein substrates bound to CNX and CRT (Molinari and Helenius
1999). The interaction between the substrate and one of the lectins
seems to be required for the interaction with ERp57. Recently the three-
dimensional structures of the CRT P-domain and the CNX ectodomain
have been solved (Ellgaard et al. 2001, 2002; Schrag et al. 2001). A struc-
tural characterization of the binding site of ERp57 on CRT has been de-
fined by NMR studies (Frickel et al. 2002). Whether ERp57 functions as
a thiol oxidase and/or a disulfide isomerase still needs to be deter-
mined.

The lectin specificity of CNX and CRT are identical, and they can bind
to the same substrate, sometimes even simultaneously. VSV G protein,
which has two N-linked glycans, binds to CNX but not to CRT (Ham-
mond et al. 1994). In contrast, Influenza HA associates with both lectins,
albeit through different glycans: CRT binds preferentially with earlier
folding intermediates and CNX associates also with a native monomeric
form of the HA (Hebert et al. 1997). Such differences between CNX and
CRT may reflect differential accessibility of the glycans in relation to the
ER membrane, because CNX has a transmembrane anchor and CRT is a
soluble protein (Danilczyk et al. 2000).

184

C. Maggioni • I. Braakman

2.2.3

Disulfide Bond Formation

Many proteins that fold in the ER contain disulfide bonds, which are re-
quired for the protein's folding, stability, and function. Disulfide bond
formation starts during synthesis; during the folding process, both na-
tive and nonnative disulfide bonds may form. One of the special features
of the ER is its oxidizing environment, similar to the extracellular space.
The formation and isomerization of disulfide bonds is catalyzed by pro-
tein thiol- disulfide oxidoreductases in the ER. The activity of this class
of proteins depends on a pair of cysteines arranged in a Cys-X-X-Cys
motif that has become a hallmark of all proteins involved in the forma-
tion or breakage of disulfide bonds. PDI was one of the first identified
proteins belonging to this class of enzymes, and it can catalyze the for-
mation, reduction, or isomerization of disulfide bonds, depending on
the redox environment (Noiva 1999; Freedman et al. 2002).

The ER needs an oxidizing milieu to be able to support disulfide
bond formation. Oxidized glutathione (GSSG) originally was thought to
be responsible for generation and maintenance of the redox conditions
in the ER, because of the higher ratio of oxidized to reduced glutathione
in the ER (1:3) compared with the cytosol (1:60) (Hwang et al. 1992).
Glutathione's assumed role, however, was completely changed by the
identification of the Erolp protein in yeast (Frand and Kaiser 1998;
Pollard et al. 1998) and Erol-La and -(5 in mammalian cells (Cabibbo et
al. 2000; Pagani et al. 2000). This protein binds to the main oxidoreduc-
tase PDI (Frand and Kaiser 1999; Benham et al. 2000;) and drives disul-
fide bond formation by maintaining PDI, and possibly other PDI family
members such as Erp44 (Anelli et al. 2002), in the oxidized state neces-
sary for disulfide transfer (Tu et al. 2000).

In yeast, electrons flow from the oxidized substrate via PDI to Erolp
to FAD, with molecular oxygen as final electron acceptor (Fig. IB, inset)
(Tu and Weissman 2002). Mammalian Erol has not been found to bind
FAD (yet), and downstream electron acceptors still need to be identified.
A second, Erol -independent, pathway for disulfide formation is pro-
posed to involve a small ER oxidase known as Erv2, a 22-kDa protein
that is noncovalently bound to FAD (Gerber et al. 2001; Sevier et al.
2001). The exact role of Erv2 in disulfide bond formation still remains
unclear (Tu and Weissman 2002). In vaccinia virus, three cytoplasmic
thiol oxidoreductases were identified that comprise a complete pathway
for disulfide bond formation for viral proteins in the relatively reducing
cytosol. Interestingly, the upstream component, El OR, is an Erv-like

Synthesis and Quality Control of Viral Membrane Proteins 185

protein. These redox proteins are conserved in all poxviruses (Senkevich
et al. 2002).

Disulfide bonds are usually highly conserved and critical for folding.
They contribute to the stability of folding intermediates perhaps even
more than to the stability of folded native proteins. Folding and disulfide
bond formation coincide, meaning that folding can be followed by as-
saying formation and isomerization of disulfide bonds, for instance,
through pulse-chase experiments (Braakman and Herbert 1996). Almost
everything we know about the role of cysteines and disulfide bonds in
protein folding comes from studies on viral proteins. The redox condi-
tions in the ER can be easily manipulated by addition of DTT in the cul-
ture medium (Alberini et al. 1990; Braakman et al. 1992): Disulfide bond
formation is inhibited, but the effect is reversed when DTT is removed.
This allowed postponing of disulfide bond formation until after synthe-
sis: completely posttranslationally. Cotranslational folding did increase
efficiency of folding but turned out to be not essential, at least not in an
intact cell where all ER factors are available.

Mutagenesis of cysteines involved in disulfide bonds has often been
applied to study their influence on folding, maturation, and intracellular
transport. Often, deletion of one cysteine residue from a pair is more
deleterious than removal of both because a single free cysteine is very
reactive and can interfere with other cysteines normally involved in oth-
er disulfide bonds. The fact that highly variable viral envelope proteins
contain completely conserved sets of cysteine residues, and therefore
most likely highly conserved disulfide bonds, demonstrates the impor-
tance of these covalent links for these proteins' functions.

Quality Control
3.1

Retention in the ER

During folding, proteins are subjected to a quality control machinery
that allows exit from the ER only for proteins that have reached the cor-
rect three-dimensional structure. Misfolded and incompletely assembled
proteins are retained in the ER and eventually degraded. The association
of newly synthesized proteins with resident ER folding factors not only
provides assistance during folding but also represents the main mecha-
nism for quality control, both at the co- and posttranslational level.

186

C. Maggioni • I. Braakman

Folding factors are indeed localized in the ER, and association with them
results in retention of the folding substrate in the ER until proper folding
is reached (Ellgaard et al. 1999). Some incompletely assembled sub-
strates can also be retained in the ER through exposure of a more specif-
ic feature. An example of this is thiol-mediated retention, which was ob-
served first for the immunoglobulin IgM (Sitia et al. 1990). During
oligomerization, the exposure of a cysteine residue in the tailpiece (cys
575), involved in an interchain disulfide bond in the polymer, is a signal
for ER retention as well as for degradation (Fra et al. 1993).

For glycoproteins, the CNX/CRT cycle is a common quality control
system, which allows the protein multiple chances to fold correctly. The
exit of certain glycoproteins from the ER to the Golgi complex is assisted
by another membrane-bound lectin, ERGIC-53, that recognizes man-
nose residues (Hauri et al. 2000). In addition to cellular factors, proper
folding of viral proteins may require complex interactions with neigh-
boring viral proteins (see Sect. 2.2; Braakman and van Anken 2000).
Many viral envelopes consist of highly ordered scaffolds of strongly in-
teracting structural proteins. These regular interactions may require
more stringent conformations for the structural proteins, resulting in
perhaps a more stringent quality control compared with the quality con-
trol of cellular proteins. It is not easy to conclude this from available
data, because of the limited set of proteins for which folding and quality
control have been studied. Actually, viral glycoproteins frequently serve
as model proteins for studies on protein folding in the ER (Doms et al.
1993). Not only for viral but also for cellular proteins, large variations
exist concerning retention in the ER: For example, 50% of newly synthe-
sized wt cystic fibrosis transmembrane conductance regulator (CFTR)
protein is degraded (Ward and Kopito 1994), whereas mutants of the
low-density lipoprotein (LDL) receptor often leave the ER to reach the
plasma membrane (Hobbs et al. 1992).

3.2

Degradation and the Unfolded Protein Response

Prolonged retention of misfolded and incompletely folded proteins in
the ER leads to their degradation (ER-associated degradation, or ERAD)
(Brodsky and McCracken 1999). This process involves the translocon,
cytosolic proteasomes, and ER chaperones such as CNX, BiP, and PDI
(Zhang et al. 2001; Molinari et al. 2002). The substrate to be degraded is
retrotranslocated through the Sec61 channel into the cytosol, deglycosy-
lated by a cytosolic N-glycanase, and, often, polyubiquitinated before

Synthesis and Quality Control of Viral Membrane Proteins 187

proteasomal degradation (Tsai et al. 2002). The criteria that determine
how proteins are identified and sent to the degradation machinery are
not completely understood. In eukaryotes, misfolded proteins are recog-
nized by the enzyme mannosidase I, which removes the terminal man-
nose residue, producing the sugar moiety Man8GlcNAc2. The Man 8 spe-
cies (including GkiMan 8 ) are recognized by a lectin, which is related to
a-mannosidase but lacks enzymatic activity (called Htmlor Mnll in
yeast and EDEM in mammals) (Jakob et al. 2001; Nakatsukasa et al.
2001). The lectin is thought to target the protein to the retro transloca-
tion pathway. Inhibition of mannosidase I, or deletion of the lectin, sta-
bilizes some misfolded glycoproteins. Which mechanism targets nongly-
cosylated proteins is still not known.

Together the CNX/CRT cycle and the ER mannosidase allow incor-
rectly folded proteins multiple chances over prolonged periods of time
to acquire the correct conformation. To handle the accumulation of in-
correctly folded proteins in the ER, however, a signaling machinery has
evolved that communicates to the nucleus that protein expression needs
to be modulated to alleviate cellular stress, referred to as the unfolded
protein response (UPR) (Ma and Hendershot 2001; Patil and Walter
2001; Harding et al. 2002). The result is an upregulation of genes encod-
ing ER folding factors. Studies in S. cerevisiae have shown that the
processes of UPR and ERAD are functionally linked to each other
(Friedlander et al. 2000; Travers et al. 2000). In this way, cells can at the
same time increase ER protein folding capacity and reduce ER protein
folding load.

It is not surprising that viral infection induces cellular stress and the
heavy load of viral protein that needs to be handled leads to an upregu-
lation of ER. However, it remains unclear whether it is the high viral ac-
tivity within the cell or the accumulation of viral proteins that induces
stress. Two examples of viruses inducing UPR are HCVand Japanese en-
cephalitis virus (JEV). HCV replication induces ER stress with the acti-
vation of UPR through transcription factor ATF6 and increased tran-
script levels of BiP (Tardif et al. 2002). During JEV infection, the lumen
of the ER rapidly accumulates substantial amounts of viral proteins that
trigger the UPR with the activation of CHOP/GADD153, a distinctive
transcription factor often induced by UPR (Su et al. 2002).

188

C. Maggioni • I. Braakman

How Viruses Evolve to Fool the Host

For a productive infection, viruses need to reproduce as many particles
as possible. They can subvert the host cell translation apparatus, affect-
ing several steps in the process of protein synthesis: degradation of host
mRNA, competition for the host translation apparatus, changing the
specificity of the host translation apparatus. In some cases, cellular pro-
tein synthesis is almost completely shut off, but this becomes rapidly
toxic to the cell. A more limited inhibition of cellular functions usually
is preferred to allow a long and productive infection.

When virus production is ensured in an infected cell, the next prob-
lem is to escape the organism's bodyguards. The immune system
evolved to counterattack viral infections, but viruses have the possibility
to evolve more rapidly and they have developed many tricks to evade
immune surveillance. The immune system is mainly based on humoral
and cellular responses, and both are targets for viral evasion.

The humoral response is based on recognition of viruses and viral
proteins in solution. Envelope glycoproteins, being on the viral surface,
are very immunogenic. Mammalian viruses therefore are subjected to
tremendous selective pressures to continually change their molecular
profiles. In many cases, natural selection produces viral strains that vary
considerably in the antigenic regions of their spike proteins. The main
strategy to mask the envelope glycoproteins from the immune system is
by antigenic variation. RNA viruses are far more susceptible to genetic
variation than DNA viruses. Three main mechanisms have been de-
scribed: (1) point mutations (antigenic drift), (2) recombination, and (3)
reassortment (antigenic shift).

1. RNA viruses lack a proofreader for replication, allowing the virus to
mutate rapidly and frequently. RNA polymerases are at least 1,000-
10,000 times more prone to error than DNA polymerases, resulting in
higher mutation rates for RNA viruses. Hot spots for mutation often oc-
cur in the viral genome, coinciding in particular with antigenic sites
recognized by virus-neutralizing antibodies. Influenza and HIV virus
are two well- characterized examples of viruses that use this strategy.
Another attempt to mask the viral envelope proteins to the humoral im-
mune system is by using host glycosylation as a means to cover or
change potential antigenic epitopes. Most viral envelope glycoproteins
are heavily glycosylated, and glycosylation sites are easily added or
deleted during evolution through antigenic drift. HIV is an example of a
virus with a highly glycosylated envelope protein (-30 glycans). In this

Synthesis and Quality Control of Viral Membrane Proteins 189

case it is not the position of the glycans in the protein but rather the
gross number of glycans that is conserved. For folding of a glycopro-
tein, the exact position is less important than the presence per se of a
glycan in that region of the polypeptide chain. Moving a glycan over
the surface of the protein often does not affect folding and maturation
but does effect shielding of antigenic sites, or disappearance of an anti-
genic epitope if the glycan was part of that epitope. It is possible that
the virus maintains the number of glycans because they indeed help the
virus to pass through cells and body unobserved.

2. Viruses can undergo recombination, during which genetic material is
exchanged with related viral or cellular sequences through cutting and
splicing of nucleic acids. This leads to a sudden change of the expressed
proteins. This feature is prominent in virus families that contain posi-
tively stranded RNA, such as the Picornaviridae and the Coronaviridae.

3. Finally, reassortment of the individual pieces of RNA may occur during
a mixed infection. For influenza virus, which does not show recombina-
tion as defined in (2), this frequently occurring antigenic shift is some-
times called recombination. Reassortment, however, leads to the pro-
duction of a virus encoding proteins of different origin. This is evident
in viruses in which the mRNA genome is present in multiple different
segments that can be exchanged without any need to cut and splice nu-
cleic acids. Other virus families with multiple genome segments include
the Rotaviridae and the Bunyaviridae. This mechanism is postulated to
account for the major antigenic shift of influenza A and is responsible
for epidemic outbreaks of this virus.

Using a cellular response, the immune system not only recognizes
virions in solution but also infected cells. Like cellular proteins, intracel-
lular viral proteins are degraded and the resulting peptides are presented
by specific molecules on the cell surface. The T cell receptor, present on
T cells, recognizes the antigen-presenting molecules MHC class I and II
associated with peptide. Recognition of a foreign antigen will result,
through different mechanims, in cell death. Almost every step in the an-
tigen presentation pathway (by both MHC I and II) has been taken as a
target for viral subversion (for complete reviews, see Tortorella et al.
2000; Vossen et al. 2002).

We will describe here only the viral products that interfere with
mechanisms related to the secretory pathway. Most examples involve the
MHC class I presentation route. First, peptides are produced through
proteasomal degradation of viral proteins. Some viral products (EBNA 1
of Epstein-Barr virus, for example) avoid their own degradation by in-
sertion of Gly-Ala repeats in their sequence. These repeats work as cis-

190

C. Maggioni • I. Braakman

acting inhibitors of proteasomal proteolytic enzymes (Levitskaya et al.
1997; Leonchiks et al. 2002). Once peptides are formed, they need to be
transferred into the ER through a channel, formed by the MHC-encoded
peptide transporter TAP. The ER-resident protein tapasin is associated
with TAR Some viruses produce a protein that interacts with the cyto-
solic part of TAP (such as herpes simplex virus ICP47; York et al. 1994);
others, such as human cytomegalovirus US6, interact with TAP on the
ER luminal face (Hengel et al. 1997; see also the chapter by Dohner and
Sodeik, this volume). Binding of US6 to TAP stabilizes a conformation
of TAP that is unable to bind ATP and hence cannot translocate peptide
(Hewitt et al. 2001). US6 itself does not contain a retention signal to sta-
bly reside in the ER, but it was suggested to bind to calnexin, through
which it would be retained in the ER (Hengel et al. 1997).

After assembly of MHC class I with peptide the complex is normally
transported to the cell surface, but many virus products can cause its in-
tracellular retention. MHC class I can be retained in the ER, for example,
by binding with the adenoviral product E3-19k (Cox et al. 1991), which
in addition binds to TAP and acts as a competitive inhibitor of tapasin
(Bennett et al. 1999). Retention can also occur in the ERGIC/cis-Golgi
compartment by binding with the murine cytomegalovirus (MCMV)
protein gp40 (ml52), which has a retention signal (Ziegler et al. 1997).
Two other proteins, gp48 (m06) and gp34 (m04), made by the same
MCMV, interfere with MHC-I molecules. gp48 directs class I molecules
to the lysosomes, whereas gp34 interacts with MHC-I in the ER; in addi-
tion, the gp34-class I complex is found on the cell surface. The exact
function of gp34 is not completely understood. It may function to si-
lence the NK response and/or alter the interaction with CD8+ T cells
(Holtappels et al. 2000).

HCMV uses other tricks to prevent MHC-I from reaching the plasma
membrane: The US3 protein retains MHC-I in the ER by a still-unknown
mechanism. The same US3 can also bind to MHC II reducing their asso-
ciation with invariant chain (Ii) (Hegde et al. 2002). Another HCMV
gene product, US2, is implicated in degradation of two important pro-
teins of MHC class II (Tomazin et al. 1999) and, together with US11, effi-
ciently directs class I molecules for proteasomal degradation with a half-
time of less than 1 min. These viral studies also showed the first example
of what turned out to be a more general process, where misfolded pro-
teins in the ER need dislocation into the cytosol to be degraded by the
proteasome (Wiertz et al. 1996; van der Wal et al. 2002).

HIV also interferes with MHC-I through two gene products: Nef and
Vpu. Nef accelerates endocytosis of MHC-I (Schwartz et al. 1996) and

Synthesis and Quality Control of Viral Membrane Proteins 191

CD4 (Rhee and Marsh 1994). The MHC-I-Nef complex accumulates in
the TGN and is then delivered to lysosomes. The mechanism of endocy-
tosis for MHC-I and CD4 is different. Endocytosis of CD4 involves clath-
rin-coated pits (Piguet et al. 1998; Williams et al. 2002), whereas MHC-I
was recently connected to the ARF6 endocytic pathway (Blagoveshchen-
skaya et al. 2002; see also the chapter by Dohner and Sodeik, this vol-
ume). Another HIV protein, Vpu, induces destabilization of newly syn-
thesized MHC-I and induces proteasomal degradation of newly synthe-
sized CD4 by phosphorylation of the protein on two specific residues
(Fujita et al. 1997; Kerkau et al. 1997; Paul and Jabbar 1997).

Conclusions

Viruses have evolved into enormously efficient infectious agents: they
exploit the cellular machinery to translate and fold their proteins and
they keep their host alive for as long as possible to allow production of
the maximum amount of progeny virus. Studies of every strategy that
viruses have developed to survive increase our knowledge of the cellular
and immunological processes involved in these strategies and will im-
prove the chances of developing immunotherapies and other therapies
for the treatment of viral infections.

Acknowledgements We thank Marije Liscalijet and Eelco van Anken for critical read-
ing of the manuscript and the audio visual department, especially Aloys Lurvink, for
assistance with the figure.

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Receptor Modulation in Viral replication: HIV, HSV,
HHV-8 and HPV: Same Goal, Different Techniques
to Interfere with MHC-I Antigen Presentation

V. Piguet (IS)

Department of Dermatology and Venerology, HUG, 24 Rue Micheli-du-Crest,

1211 Geneva, Switzerland

vincent.piguet@medecine.unige.ch

1 Introduction 200

2 Receptor Modulation During Viral Replication:

Interference with MHC-I Antigen Presentation 201

2.1 Primate Lentiviridae Interference with MHC-I Presentation:

Connecting the Receptor with the PACS-1 -Dependent Pathways 201

2.2 HSV Downregulation of MHC-I Expression:

Targeting the Transporter TAP 205

2.3 HHV-8 Downregulation of MHC-I Expression:

Ubiquitination of the Tail 206

2.4 HPV Inhibition of MHC-I Expression: Blocking the Proton Pump. ... 210

3 Perspectives 211

References

212

Abstract Evasion of host immunity is a common objective of viruses that cause
chronic infections. Viruses involved in sexually transmitted infections constitute no
exception to this phenomenon. HIV, HPV, HSV, and HHV-8 subvert the class I major
histocompatibility complex (MHC-I) antigen presentation pathway, thereby evading
the cellular immune response. Although the goal of these viruses is the same and ef-
ficient MHC-I downregulation in infected cells is achieved, their techniques vary
considerably. Whether viral inhibition occurs at the transcriptional level, during as-
sembly of MHC-I complexes in the endoplasmic reticulum, during its journey to the
cell surface, or after reaching the cell surface, each one of these viruses ingeniously
achieves MHC-I downregulation and avoids the cellular immune response. Unravel-
ing the mechanisms of interference with MHC-I antigen presentation employed by
these viruses is not only crucial to understand their pathogenesis, but also reveals
novel mechanisms of regulation of cellular receptors. When employed as modulators
of cellular trafficking pathways, viruses become tools to dissect fundamental cell
processes. In return, the precise dissection of these processes may offer new weap-
ons against the ruses viruses employ to propagate and establish chronic infections.

200

V. Piguet

Introduction

Cells require surface receptors for the capture of essential constituents
and energy, for adaptation to their environment via specific signaling
cascades, and for displaying information that is read by other cells or by
extracellular factors. Surface receptors are at the core of the interactions
that take place between a cell and the environment in which it resides.
As a result, the expression of most cell surface receptors is tightly regu-
lated through specialized processes (for recent review see Conner and
Schmid 2003). Viruses often modulate the expression of cell surface re-
ceptors, in order to maximize their replication and survival in an infect-
ed host. A typical example is the modulation of class I major histocom-
patibility complex (MHC-I) surface levels by viruses that establish
chronic infections, in order to avoid the cellular arm of the immune re-
sponse. This review will focus on the mechanisms of regulation of
MHC-I antigen presentation by viruses implicated in sexually transmit-
ted infections, in other words: HIV, herpes simplex virus (HSV), human
herpesvirus 8 (HHV-8), and HPV.

MHC-I consists of a highly polymorphic, membrane-anchored heavy
chain noncovalently associated with /?2 -microglobulin (film.). The as-
sembly of the heavy chain with film takes place in the ER or in the cis-
Golgi apparatus, where antigenic peptides are loaded (Bijlmakers and
Ploegh 1993). A significant fraction of newly synthesized proteins, viral
or cellular, undergoes proteasomal degradation generating peptides
(Schubert et al. 2000). Antigenic peptides are supplied by the trans-
porter associated with antigen processing, TAP. Without its peptide car-
go, Class I complexes do not reach the cell surface, are unstable, and dis-
sociate. The trimolecular complex (heavy chain with /?2m and peptide)
is subsequently addressed to the cell surface (Neefjes et al. 1990). MHC-I
complexes are stably expressed at the cell surface, with only a minor
fraction of the molecules being internalized spontaneously in T cells and
in monocytes/macrophages (Neefjes et al. 1990; Reid and Watts 1990).
In an attempt to paralyze the cellular immune response, viruses are able
to target MHC-I at almost all steps of its trafficking: in the ER, on its
way to the surface, and after it reaches the cell surface.

Receptor Modulation in Viral replication: HIV, HSV, HHV-8 and HPV 201

Receptor Modulation During Viral Replication:
Interference with MHC-I Antigen Presentation

2.1

Primate Lentiviridae Interference with MHC-I Presentation:
Connecting the Receptor with the PACS-1 -Dependent Pathways

The human and simian immunodeficiency viruses (HIV and SIV) are
lentiviruses, members of the retrovirus superfamily characterized by
their genomic complexity. Besides the Gag, Pol, and Env genes found in
all retroviruses, HIV and SIV each contain six additional reading frames.
Two of these, Tat and Rev, encode crucial regulators of viral gene
ex-pression. The four others, Nef, Vif, Vpr, and Vpu (in HIV-1) or Vpx
(in HIV-2 and SIV), are dispensable for viral growth in most tissue cul-
ture systems. As a consequence, these genes are termed auxiliary or ac-
cessory. However, the so-called accessory proteins of primate lentivirus-
es represent critical virulence factors that manipulate several cellular
pathways in order to maximize viral replication, including downregula-
tion of MHC-I complexes from the cell surface. HIV can, at least to some
extent, escape the attacks of cytotoxic T lymphocytes (CTLs) by down-
regulating the expression of MHC-I on the surface of infected cells in
vitro and in animal models (Collins et al. 1998; Munch et al. 2001;
Schwartz et al. 1996). This manipulation of the cellular immune re-
sponse might enable HIV to persist in the host for prolonged periods of
time, which leads ultimately to a collapse of the immune system called
acquired immunodeficiency syndrome (AIDS).

The main viral player that interferes with MHC-I antigen presentation
is Nef (Schwartz et al. 1996; Fig. 1), even though two other viral proteins,
Vpu and Tat, may occasionally play a minor role (Howcroft et al. 1993;
Kerkau et al. 1997). Nef is a short cytoplasmic protein that associates
with membranes through N-terminal myristoylation and exerts several
additional effects on the infected cell. For instance, it is responsible for
downregulating the cell surface expression of CD4, the main HIV recep-
tor (Garcia and Miller 1991). Nef downmodulates MHC-I in a variety of
cell types, including primary T lymphocytes (Kasper and Collins 2003;
Mangasarian et al. 1999; Schwartz et al. 1996). Determinants necessary
for Nef responsiveness are contained in the cytoplasmic domain of
MHC-I and are centered around a critical tyrosine residue found in
HLA-A and -B, but not in HLA-C (Le Gall et al. 1998). Correspondingly,
HLA-C is unaffected by Nef (Cohen et al., 1999). This may be of physio-

202

V. Piguet

Early @ndo$ome&

Late endosomes

Lysosomes

PACS-1 (blue) + AP-1 (green)
Clathrin

Fig. 1. HIV Nef connects MHC-I to the PACS-1 sorting pathway. HIV Nef interacts
with MHC-I cytosolic domains and connects them to the PACS-1 sorting pathway. 1
Nef is first targeted to the frarcs-Golgi network (TGN) via its interaction with PACS-1
(phosphofurin acidic cluster sorting protein), where it activates phosphatidylinositol
3-kinase (PI3K*) via its polyproline domain (Nef*). 2 In turn, PI3K activates ADP ri-
bosylation factor 6 (ARF6*). 3 Nef interacts with MHC-I cytoplasmic domain and
redirects MHC-I from the cell surface to the ARF-6 specific early endosomal com-
partment. 4 MHC-I is then redirected to the TGN by Nef via its acidic cluster domain
and PACS-1 recruitment

logical importance, because HLA-C molecules are dominant inhibitory
ligands that protect cells against lysis by natural killer (NK) lympho-
cytes, which normally destroy MHC-I -negative cells (Cohen et al. 1999).
Recently, Nef has also been shown to interact with HLA-A and B both in
vitro and in vivo, but not with HLA-C (Williams et al. 2002).

In the presence of Nef, MHC-I is normally synthesized and transport-
ed through the ER and cis- Golgi and can reach the cell surface. However,
it is then diverted toward the endosomal pathway to accumulate in the
trans-Golgi network (TGN), before undergoing degradation (Greenberg
et al. 1998b; Le Gall et al. 1998; Schwartz et al. 1996). Some degree of in-
terference also occurs during MHC-I transport from the TGN to the cell
surface (Kasper and Collins 2003). Mutagenesis studies have highlighted
the importance of specific determinants in the ability of HIV-1 Nef to
regulate MHC-1. These include the Nef myristoylation signal, essential

Receptor Modulation in Viral replication: HIV, HSV, HHV-8 and HPV 203

for its membrane association, an N-proximal a-helix, a centrally-located
SH3 -binding proline-based repeat, and a highly conserved cluster of
acidic residues in the N-terminal third of the viral protein (EEEE 65 )
(Greenberg et al. 1998b; Mangasarian et al. 1999). The acidic cluster
(AC) of Nef is highly similar to the phosphofurin acidic cluster sorting
protein- 1 (PACS-l)-binding, TGN retrieval motif of furin and mannose-
6-phosphate receptor (MPR). PACS-1 governs the endosome-to-Golgi
trafficking of furin and MPR by connecting the AC-containing cytoplas-
mic domain of these molecules with the adaptor protein complex (AP-1)
of endosomal clathrin-coated pits (CCP) (Wan et al. 1998). Likewise, the
AC-mediated recruitment of PACS-1 is responsible for MHC-I downreg-
ulation and TGN targeting (Crump et al. 2001; Piguet et al. 2000). Nef
and PACS-1 interact to hijack the ADP ribosylation factor 6 (ARF6)-de-
pendent endocytic pathway, by a process involving phosphatidylinositol
3-kinase (PI3K), leading to a loss of cell surface MHC-I. This mechanism
requires the sequential actions of three Nef motifs. The acidic cluster
EEEE 65 , the SH3 domain binding proline-based repeat, and a N-proxi-
mal a-helix are required one after the other in controlling PACS-1 -de-
pendent sorting to the TGN, ARF6 activation, and sequestration of inter-
nalized MHC-I to the TGN, respectively. As expected, inhibitors of PI3K
block retrieval of MHC-I molecules to the TGN (Blagoveshchenskaya et
al. 2002). The most likely route taken by MHC-I in Nef-expressing cells
is the following: First, Nef is targeted to the TGN via its interaction with
PACS-1, where it activates PI3K via Nef proline-based repeat. Second,
PI3K activates ARF6. Third, Nef interacts with MHC-I cytoplasmic do-
main and redirects MHC-I from the cell surface to the ARF6 specific ear-
ly endosomal compartment. Fourth, MHC-I is sent to the TGN by Nef
via its AC domain and PACS-1 recruitment. Finally, MHC-I is retained in
the TGN, via the N-proximal a-helix of Nef. Together, these results sup-
port a model in which Nef downregulates MHC-I by acting as a connec-
tor between the receptor cytoplasmic tail and the PACS-1 sorting path-
way. However, a direct demonstration of complexes containing MHC-I,
Nef, and PACS-1 in cells remains to be shown.

Nef acts also as a connector to downregulate CD4, the main HIV re-
ceptor. However, in this case both the downstream partners of Nef and
the fate of its cellular target are different. In the Golgi and at the plasma
membrane, Nef bridges the cytoplasmic tail of CD4 with the adaptor
protein complex of CCP, thereby triggering the formation of CD4-specif-
ic endocytic vesicles. Then, in the early endosome, Nef links CD4 to a
subset of COP-I coatomer proteins that then target the HIV receptor for
degradation in lysosomes (Aiken et al. 1994; Greenberg et al. 1997; Le

204

V. Piguet

Gall et al. 2000; Mangasarian et al. 1997; Piguet et al. 1998; Piguet et al.
1999; Rhee and Marsh 1994).

Interestingly, Nef can also recruit the catalytic sub unit H of the vacu-
olar membrane ATPase (V-ATPase), which may facilitate CD4 internal-
ization and the recruitment of components of CCP (Geyer et al. 2002b;
Lu et al. 1998). A further effect of Nef-induced CD4 downregulation is to
contribute to the fitness of viral replication, because it preserves the in-
fectivity of HIV- 1 virions by preventing interference between the recep-
tor and particle release or envelope incorporation (Lama et al. 1999;
Ross et al. 1999).

Interestingly, although Nef downregulates both CD4 and MHC-I, it
can increase the surface levels of a dendritic cell-specific receptor called
DC-SIGN. Nef inhibits the endocytosis of DC-SIGN (Sol-Foulon et al.
2002), thus increasing the surface levels of this receptor. DC-SIGN facili-
tates infection of T cells in trans and is also responsible for the interac-
tion between dendritic cells and naive T cells (Geijtenbeek et al. 2000a,
2000b). The mechanism of Nef inhibition of DC-SIGN endocytosis is
partly elucidated. DC-SIGN contains a sorting signal characterized by a
dileucine domain in its cytoplasmic domain (Engering et al. 2002). This
domain is required for DC-SIGN sorting to the lysosomes in immature
dendritic cells. Nef also requires its own dileucine domain to inhibit DC-
SIGN endocytosis. The dileucine motif of Nef is required to interact with
adaptor complexes at the plasma membrane (Bresnahan et al. 1998;
Craig et al. 1998; Greenberg et al. 1998a). The simplest explanation of
Nef-induced DC-SIGN upregulation is that Nef interferes with DC-SIGN
sorting pathways because of a direct competition between Nef dileucine
domain and DC-SIGN dileucine motif, both interacting with similar
adaptor complexes at the plasma membrane. The consequence of Nef-
mediated upregulation of DC-SIGN is an increase in viral replication in
dendritic cell-Tcell clusters (Sol-Foulon et al. 2002).

Manipulation of the surface levels of cellular receptors by HIV is
complex but offers novel possibilities to understand the mechanisms
that this virus employs to persist in the host. For instance, by under-
standing the precise role of Nef in enhancing viral replication and avoid-
ing the cellular immune response, drugs could be developed that target
domains in Nef involved in MHC-I downregulation. A potential benefit
of Nef inhibitors would be an increase in viral recognition by the im-
mune system and a decrease in viral replication.

Receptor Modulation in Viral replication: HIV, HSV, HHV-8 and HPV 205

2.2

HSV Downregulation of MHC-I Expression: Targeting the Transporter TAP

Herpes simplex virus (HSV) infections are the source of recurrent muco-
cutaneous infection, such as herpes labialis and herpes genitalis. Occa-
sionally HSV infections are the cause of life-threatening or sight-impair-
ing disease, especially in neonates and the immunocompromised patient
population. After primary infection, the virus persists for life in a latent
form in neurons of the host. Again, interference with the MHC-I antigen
presentation pathway may play a central role in HSV persistence. Unlike
HIV Nef, which targets MHC-I after its exit from the ER, HSV types 1 and
2 express an early gene product, ICP47, which retains MHC-I complexes
in the ER (York et al. 1994; see also the chapter by Maggioni and Braak-
man, this volume). ICP47 is a cytosolic protein that inhibits the function
of TAP by interacting stably with its cytosolic domain (Fruh et al. 1995;
Fig. 2). This interaction blocks TAP-dependent peptide translocation into
the ER (Ahn et al. 1996; Tomazin et al. 1996). Consequently, MHC-I com-
plexes are (1) unstable, (2) retained in the ER, and (3) degraded. Indeed,
when MHC-I molecules are unable to assemble properly, the misfolded
MHC- 1 heavy chain is removed from the ER to the cy tosol, deglycosylat-
ed, and degraded by the proteasome (Hughes et al. 1997). ICP47 induces
the accumulation of misfolded heavy chains in the ER, which leads to pro-
teosomal degradation. The most likely mechanism by which ICP47 in-
hibits TAP is to act as a pseudo-substrate inhibitor. In favor of this hy-
pothesis is the fact that the N-terminal domain of ICP47 (residues 2-35)
is sufficient to interfere with TAP function (Neumann et al. 1997). Howev-
er, direct competition with the pep tide-binding domain of TAP might not
show the whole picture. Indeed, ICP47 also inhibits the ATPase activity of
TAP, which is required for peptide translocation (Gorbulev et al. 2001).
Few viruses target TAP in order to inactivate MHC-I antigen presentation.
The only other known example is the HCMV US6 protein. HCMV belongs
to a subfamily of herpesviruses distinct from HSV. Unlike ICP47, US6
does not appear to block the binding of the peptides to TAP (from the cy-
tosolic side) but inhibits the translocation of peptide into the ER (ER-lu-
minal side) (Ahn et al. 1997; Hengel et al. 1997). As a consequence, US6
disturbs the assembly of MHC-I molecules (reviewed by Momburg and
Hengel 2002). The benefit from these studies is to use viruses as tools to
dissect the function of the peptide transporter TAP, as well as potentially
identifying molecules that would block the viral protein, while leaving
TAP function unaltered. Such compounds could facilitate viral clearance,
thereby preventing persistence of herpesviruses in the host.

206

V. Piguet

Late en do some

HPVE5

Fig. 2. Blocking transporters and pumps: TAP and the V-ATPase. Blue: HSV ICP47
blocks the peptide transporter TAP (transporter associated with antigen processing)
on the cytosolic side of the ER. CMV U6 protein blocks TAP from the ER-luminal
side. Both viral proteins induce an accumulation of unstable MHC-I molecules,
which leads to their rapid degradation in the proteasome. Red: 1 HPV E5 protein de-
creases MHC-I expression at the transcriptional level. 2 HPV E5 interferes with the
V-ATPase, a proton pump. This leads to a loss of acidification of the TGN and of the
endosomes, leading to the accumulation of MHC-I in the TGN

2.3

HHV-8 Downregulation of MHC-I Expression: Ubiquitination of the Tail

Kaposi's sarcoma-associated herpesvirus (KHSV; HHV-8) is a lympho-
tropic y2-herpesvirus that is strongly implicated in the pathogenesis of
Kaposi's sarcoma and two AIDS-related lymphoproliferative syndromes:
primary effusion lymphoma and multicentric Castleman's disease (re-
viewed in Hengge et al. 2002). DNA sequence analysis of the HHV-8 ge-
nome revealed, in addition to a number of homologs of cellular proteins
[including a virus-encoded interleukin-6, MlPl-a and -(5 chemokines,
Bcl-2, dihydrofolic reductase, and thymidylate synthetase (Nicholas et
al. 1997)], several unique open reading frames, K3, K4.2, K5, and K7,
that do not share homology with any known cellular genes (Nicholas et
al. 1997; Russo et al. 1996). Interestingly, K3 and K5 [otherwise known
as modulator of immune recognition (MIR)l and MIR2] display 40%

Receptor Modulation in Viral replication: HIV, HSV, HHV-8 and HPV

207

Proteasome

Fig. 3. Ubiquitination of receptor cytoplasmic tails. Red: 1 KHSV MIR1 and MIR2
induce the ubiquitination of MHC-I cytoplasmic domain, thereby inducing its rapid
endocytosis via a clathrin dependent pathway. MIR1 and -2 function directly as E3
ubiquitin ligases. 2 MHC-I is then rerouted to the TGN and finally (3) degraded in
the lysosomes. TSG101 is required for this last step (3). Blue: HIV VPU induces
ubiquitination of CD4 cytoplasmic domain in the ER by acting as a connector be-
tween the receptor cytoplasmic tail and h-ySTrCP, a protein that provides a link with
the ubiquitin proteolysis machinery. Ubiquitinated CD4 is then is sent to the protea-
some for destruction

amino acid homology with each other (Russo et al. 1996) and are ex-
pressed during the early lytic cycle of viral replication. MIR1 and -2 en-
code transmembrane proteins that downregulate cell surface MHC-I, as
well as ICAM-1 and B7-2, ligands for NK cell-mediated cytotoxicity re-
ceptors. As a consequence, K5 expression drastically inhibits both
MHC-I antigen presentation and NK cell-mediated cytotoxicity (Ishido
et al. 2000a). HHV-8 interference of MHC-I antigen presentation is me-
diated by mechanisms that involve ubiquitination of the cytosolic do-
main of MHC-I (Coscoy et al. 2001; Fig. 3). After reaching the cell sur-
face MHC-I complexes are rapidly internalized and targeted to lyso-
somes, where they are degraded (Ishido et al. 2000b). Endocytosis of
MHC-I complexes may occur via clathrin-mediated endocytosis, be-
cause it can be inhibited by a dominant-negative form of dynamin
(Coscoy and Ganem 2000). The motifs conferring sensitivity of MHC-I

208

V. Piguet

to MIR1 and MIR2 are lysines in the cytoplasmic tail of MHC-I (Coscoy
et al. 2001). Furthermore, MHC I is ubiquitinated in MIR1- and MIR2-
transfected cells and requires the presence of these lysines in the cyto-
plasmic tail of MHC-I. MIR1 and MIR2 both contain cytosolic zinc fin-
gers of the PHD subfamily. PHD motifs share both sequence and struc-
tural homology with RING finger domains that are capable of E3 ubiqui-
tin ligase activity (Joazeiro and Weissman 2000). E3 ubiquitin ligases
give specificity to the ubiquitination process by recruiting the E2 ubiq-
uitin- conjugating enzyme and directing ubiquitin onto the substrate. In-
deed, proteins containing the MIR2 PHD domain can undergo auto-
ubiquitination in the presence of El, E2, ubiquitin, and ATP in vitro,
which demonstrates that MIR2 functions as an E3 ubiquitin ligase that
relies on its PHD domain (Coscoy et al. 2001). Mutations of the zinc-co-
ordinating residues abrogate the PHD domain ubiquitin ligase activity
as well as MHC-I downregulation.

Internalization of ubiquitinated MHC-I complexes by MIR1 is fol-
lowed by the redirection of MHC-I to the TGN and finally to the lyso-
somes, where they are degraded (Means et al. 2002). Specific motifs in
MIR1 are sequentially involved to remove MHC-I from the cell surface:
An N-terminal zinc finger motif and a central sorting motif are involved
in triggering internalization of MHC-I molecules and redirecting them
to the TGN. Subsequently, the C-terminal diacidic cluster region of
MIR1 is engaged in targeting MHC-I molecules to lysosomes (Means et
al. 2002).

MIR1 targeting of MHC-I complexes to lysosomes requires another
cellular partner, TSG101 (see also the chapter by Maggioni and Braak-
man, this volume). TSG101, the mammalian homolog of yeast Vps23
(Katzmann et al. 2001), is known to function in late endosome sorting
in mammalian cells (Babst et al. 2000). Experiments using small interfer-
ing RNA (siRNA) have demonstrated that in the absence of TSG101
MIR1 is unable to target MHC-I complexes to lysosomal degradation,
strongly suggesting that the viral protein can use the TSG101 -dependent
sorting pathway (Hewitt et al. 2002).

Ubiquitination of the cytosolic domain of a cellular receptor in order
to target it to degradation is not unique to KHSV proteins MIR1 and -2.
The lentiviral protein Vpu is also capable of mediating the ubiquitina-
tion of the cytosolic domain of CD4 (and possibly MHC-I) in order to
target them to degradation. This downregulation occurs at the ER, be-
fore transport of newly synthesized proteins to the cell surface. This dif-
ferentiates Vpu from the KHSV proteins MIR1 and -2, which act mainly
after MHC-I has reached the cell surface. Vpu is a 16-kDa type I integral

Receptor Modulation in Viral replication: HIV, HSV, HHV-8 and HPV 209

membrane phosphoprotein found exclusively in HIV-1 and SIV C pz but
absent in HIV-2 and most SIV strains (Cohen et al. 1988; Strebel et al.
1988). Its 81-amino acid-long sequence encompasses an N-terminal hy-
drophobic membrane-spanning domain of 24 residues and a C-terminal
cytoplasmic tail capable of forming oligomers. Vpu is expressed on in-
tracellular membranes but not at the cell surface. Vpu induces the deg-
radation of CD4 molecules retained in the ER as CD4-Env complexes
(Willey et al. 1992). Like Nef, Vpu acts as a connector between CD4 and
cellular degradative pathways. In this case, however, the proteasome
rather than the lysosome is the entity responsible for CD4 destruction
(Schubert et al. 1998). Vpu bridges the CD4 cytoplasmic tail, which it
directly binds, with a protein known as h-/?TrCP (Margottin et al. 1998).
h-/?TrCP contains an F-box motif and seven WD repeats. The WD repeat
region of h-/?TrCP interacts with Vpu in a phosphoserine-dependent
manner while the F-box recruits Skplp, a protein that provides a link
with the ubiquitin proteolysis machinery. Whether Vpu action involves
dislocation of CD4 from the ER into the cytoplasm, direct targeting of
the cytosolic part of the glycoprotein by the proteasome, or another as
yet undefined mechanism remains unclear. However, the model in which
Vpu targets CD4 for proteosomal degradation is supported by the obser-
vation that Vpu action involves the ubiquitin pathway and is sensitive to
inhibitors of cytosolic proteasome (Fujita et al. 1997). In some systems
Vpu may interfere with MHC-I surface expression. This interference oc-
curs at an early step in the biosynthesis of MHC-I molecules (Kerkau et
al. 1997). It can be speculated that Vpu also induces the ubiquitination
of MHC-I molecules, thereby targeting them to degradation before their
arrival at the cell surface.

Ubiquitination of cellular receptors is not only a ploy used by viruses
to remove receptors from the cell surface for their own benefit, but also
a general mechanism to control surface levels of some receptors (Hicke
2001). For instance, a cellular homolog of MIR1 and MIR2 has been
identified and called c-MIR (Goto et al. 2003). c-MIR induces a specific
downregulation of B7-2 through ubiquitination, rapid endocytosis, and
lysosomal degradation, processes very similar to MIR1 and -2 from
KHSV. This is a clear example of how a process first identified as an
anecdote linked with a viral infection may reveal more general mecha-
nisms of regulation of endocytosis.

210

V. Piguet

2.4

HPV Inhibition of MHC-I Expression: Blocking the Proton Pump

Human papillomaviruses are oncogenic viruses that cause benign hy-
perproliferative lesions in the skin and mucosae (warts and condylo-
mas). These lesions tend to persist for prolonged periods of time and
may occasionally progress toward tumors. Viral persistence is required
for oncogenic transformation to occur (Tindle 2002). The papillomavi-
rus genome is approximately 7.8 kb and is divided into early, late, and
noncoding regions. At least two viral early proteins (E5 and E7) are re-
sponsible for removal of MHC-I from the cell surface. The early gene
product E7 of the highly oncogenic strains HPV- 16 and -18 downregu-
lates the MHC-I heavy chain promoter and the TAP-1 and LMP-2 pro-
moters (Georgopoulos et al. 2000), which leads to a decrease of MHC-I
expression. Another HPV protein called E5 can also downregulate
MHC-I expression at least at two levels. First, E5 decreases MHC-I ex-
pression at the transcriptional level. Second, expression of E5 causes re-
tention of MHC-I in the Golgi apparatus, thus preventing its transport
to the cell surface (Ashrafi et al. 2002; Marchetti et al. 2002). E5 there-
fore regulates MHC-I surface levels by different mechanisms, but pre-
vention of MHC-I transport to the cell surface appears to be the domi-
nant effect. The mechanisms of MHC-I retention in the Golgi are still
unclear. E5 can interact with the subunit c of the vacuolar proton pump
(V-ATPase; Goldstein et al. 1991). Furthermore, E5 expression leads to
the inhibition of the acidification of endosomes and of the Golgi appa-
ratus (Schapiro et al. 2000; Straight et al. 1995). E5 mutants that have
lost their ability to bind the proton pump are unable to modify Golgi
acidification (Schapiro et al. 2000) suggesting that E5 inhibits the V-AT-
Pase directly. Whether this block of acidification leads to unstable
MHC-I complexes that are retained in the Golgi apparatus remains to
be established. E5 binding to the V-ATPase is to be compared to HIV-
Nef interaction with the V-ATPase. In the case of E5, the viral protein
seems to inactivate the proton pump in order to affect endosomal and
TGN pH, which leads to perturbation in MHC-I trafficking. In the case
of HIV-1 Nef, no functional perturbation of the V-ATPase has yet been
identified and Nef might just use this interaction to enhance its associa-
tion with the adaptor complexes while downregulating CD4 from the
cell surface (Geyer et al. 2002a, 2002b; Lu et al. 1998). Nef expression in
cells does not modify the pH of endosomes but redirects the trafficking
of cellular receptors toward acidic compartments (Piguet et al., 1998). It
is thus conceivable that HPV E5 and HIV-Nef proteins hijack the same
cellular target, the V-ATPase, but then subvert two separate functions

Receptor Modulation in Viral replication: HIV, HSV, HHV-8 and HPV 211

of the proton pump. Further experiments comparing the activities of
the two viral proteins might provide a better understanding of the func-
tion of the V-ATPase that regulates the pH in the exo- and endocytic
pathways.

Perspectives

A common necessity for viruses that persist in the host is interference
with MHC-I antigen presentation. A common objective could imply sim-
ilar mechanisms for achieving this goal. However, viruses have evolved
complex strategies to paralyze MHC-I at all levels of its biogenesis, as-
sembly, and trafficking. HPV E5 protein blocks MHC-I expression at the
transcriptional level. HSV protein ICP47 inhibits TAP function, leading
to retention of MHC-I complexes in the ER. HPV E5 blocks the acidifica-
tion of the Golgi and of endosomes and leads to the accumulation of
MHC-I in the Golgi. HIV Nef reroutes MHC-I complexes from the sur-
face to the TGN, acting as a connector between the receptor cytoplasmic
tail and the PACS-1 sorting pathway. Finally, the KHSV proteins MIR1
and MIR2 induce the ubiquitination of MHC-I complexes, leading to
their rapid internalization from the cell surface, targeting to the TGN,
and finally the lysosomes. The careful dissection of the ploys used by
viruses to interfere with MHC-I presentation has led to the discovery of
novel mechanisms of regulation of surface receptors, which are essential
for a proper cell function, such as the PACS-1 -dependent pathway or en-
docytosis regulated by ubiquitination. Furthermore, considerable pro-
gress has been achieved in the understanding of TAP and other cofactors
necessary for the proper assembly of MHC-I complexes. Whether these
studies will enable us to develop therapeutic strategies blocking the viral
proteins that downregulate MHC-I and whether such drugs aimed at
restoring normal surface levels of MHC-I on infected cells will lead to
viral clearance remain to be demonstrated.

Acknowledgments This work was supported by the Geneva Cancer League and Swiss
National Science Foundation grant No 3345-67200.01 to VP. VP is the recipient of a
"Professor SNF" position (PP00A-68785).

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V. Piguet

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Trafficking of Viral Membrane Proteins

R. Byland • M. Marsh (is)

Cell Biology Unit, MRC-LMCB and Department of Biochemistry
and Molecular Biology, University College London, Gower Street,
London, WC1E 6BT, UK
m.marsh@ucl.ac.uk

1 Introduction 219

2 The Cellular Protein Sorting and Trafficking Machinery 221

3 Traffic of Viral Envelope Proteins 227

3.1 Retroviruses 227

3.2 Herpesviruses 233

3.3 Orthomyxoviruses 237

3.4 Rhabdoviruses 238

3.5 Poxviruses 239

4 Virus Assembly 240

4.1 ESCRTing Virus Release 240

4.2 Other Late Domain Sequences 242

5 Conclusions 243

References

244

Abstract Many viruses express membrane proteins. For enveloped viruses in particu-
lar, membrane proteins are frequently structural components of the virus that medi-
ate the essential tasks of receptor recognition and membrane fusion. The functional
activities of these proteins require that they are sorted correctly in infected cells.
These sorting events often depend on the ability of the virus to mimic cellular pro-
tein trafficking signals and to interact with the cellular trafficking machinery. Impor-
tantly, loss or modification of these signals can influence virus infectivity and patho-
genesis.

Introduction

The final steps in the assembly of enveloped viruses occur in the context
of a cellular membrane when the nascent particle undergoes a budding
reaction that simultaneously generates the viral envelope and releases

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the free virion. The cellular membrane can be the plasma membrane,
leading to virus release directly to the extracellular space, or an intracel-
lular membrane (e.g. the ER, Golgi apparatus or endosomal system), in
which case the virions are delivered into intracellular vacuoles from
which they are released to the extracellular space by a secretory-type
mechanism. During the budding process membrane proteins are incor-
porated into the viral envelope. These can include integral membrane
proteins and peripheral proteins, and both types can be virally encoded
or of cellular origin. The number of envelope proteins varies greatly be-
tween different virus families. Genetically relatively simple viruses, such
as rhabdoviruses, encode a single integral membrane glycoprotein that
mediates the key entry functions of receptor recognition and membrane
fusion. More complex viruses, such as herpesviruses, encode up to 60
putative membrane proteins, many of which can be found in the viral
envelope (Britt and Mach 1996; Spaete et al. 1994).

To ensure that fully infectious virions are produced, the components
of a mature virus must be brought together in infected cells in a tempo-
rally and spatially co-ordinated manner. The proteins must be synthe-
sized at an appropriate time and transported to the membrane system
where they will be incorporated into domains that will become viral en-
velopes. As discussed in the chapter by Maggioni and Braakman, this
volume, integral membrane proteins are synthesized on the ER and use
cellular mechanisms to ensure correct folding, quality control and ex-
port to the Golgi apparatus. Subsequently these proteins must be trans-
ported, directly or indirectly, to sites in the cell where budding occurs.
The transport events may require transit through specific cellular com-
partments where, for example, glycosylation is completed or proteolytic
cleavage occurs. For this transport, viruses exploit cellular trafficking
machineries and use signals that frequently mimic those found in cellu-
lar proteins.

Although most studies have focussed on integral membrane proteins,
similar types of processes must occur for viral peripheral membrane
proteins, such as retroviral Gag polyproteins (the precursor for viral
matrix and capsid proteins) that associate with the cytoplasmic side of
cellular membranes and are subsequently located on the interior of as-
sembled virions. These proteins are synthesized on free polysomes and
targeted to membranes by a N-terminal myristic acid moiety plus, in
many cases, a second motif such as an adjacent stretch of basic amino
acids (reviewed in Bijlmakers and Marsh 2003). For many peripheral
membrane proteins, association with a specific membrane system may
be direct, but for others targeting may involve vesicular transport. Cur-

Trafficking of Viral Membrane Proteins 221

rently little is known of the mechanisms involved in the trafficking of
these peripheral proteins.

In addition to structural proteins of the virion, a number of viruses
encode membrane proteins with other roles. These can include proteins
that modify the cell surface expression of MHC antigens or clear chemo-
kines or antibodies from the environment around infected cells, thereby
modifying the efficacy of the host's immune response to infection.
Again, these proteins use host cell trafficking machineries and signals to
effect their functions.

Here we discuss some of the molecular signals used by viruses to dis-
tribute their membrane proteins within infected cells and how these sig-
nals contribute to virus assembly and/or pathogenesis. For the most part
the discussion will focus on integral membrane proteins and reference
to peripheral proteins will be limited. In addition, we also review the
role of the cellular ESCRT machinery in the assembly of certain en-
veloped viruses.

The Cellular Protein Sorting and Trafficking Machinery

A considerable amount of information has now emerged on the traffick-
ing pathways in eukaryotic cells and the cellular and molecular mecha-
nisms through which these pathways operate. The vacuolar apparatus
can be considered as a series of functionally overlapping membrane-
bound compartments:

- The ER/Golgi systems, in which glycoprotein synthesis, folding,
oligomerization, glycosylation, acylation and quality control occur

- The TGN/endosomal systems, in which many of the sorting reactions
that control constitutive and regulated secretion, polarity and endocy-
tosis occur

- The plasma membrane

- The late endosomal/lysosomal systems, in which many receptors and
their ligands, plus other membrane proteins and material internalized
by endocytosis, are degraded

A network of trafficking pathways (see Fig. 1) mediates transport be-
tween the different compartments in a highly regulated manner (Boni-
facino and Glick 2004; Bonifacino et al. 1996; Hirst and Robinson 1998;
Pelkmans and Helenius 2002; Sorkin 2000). After synthesis, the default

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R. Byland • M. Marsh

Lipid raft

Fig. 1. Cellular endocytic and exocytic trafficking pathways. Arrows depict known
transport pathways between different organelles. Where known, relevant coat and
adaptor proteins are indicated in boxes. Multivesicular body (MVB) formation as an
example of an outwardly directed vesiculation process is indicated (*)

pathway for secretory proteins leads them from the ER to the Golgi appa-
ratus and then via the TGN to the plasma membrane. Many plasma
membrane proteins follow a similar route. Some proteins, for example
the cation-independent mannose-6-phosphate receptor (CI-M6PR), that
carries lysosomal enzymes, are directed from the TGN to late endosomes
via early endosomes (Kornfeld and Mellman 1989; Tikkanen et al. 2000;
Trowbridge et al. 1993). Proteins delivered to the plasma membrane can
be reinternalized by clathrin-dependent or clathrin-independent/lipid
raft-dependent pathways to endosomal organelles from where they may
be recycled, targeted to lysosomes or sorted to other cellular compart-
ments including the Golgi apparatus, ER or alternative plasma membrane
domains. In polarized cells, membrane and secretory proteins can be tar-
geted to either basolateral or apical domains and sorting to these do-
mains can occur at the TGN or in endosomes. Some of the signals in-
volved in these latter sorting steps are related to endocytosis signals and
appear to be interpreted by similar machineries. These pathways may
also exist in non-polarized cells (Scheiffele et al. 1997), although epithe-
lial-specific sorting reactions do occur (Sugimoto et al. 2002).

Trafficking of Viral Membrane Proteins 223

The trafficking of membrane proteins in the vacuolar apparatus in-
volves sorting signals and a set of cellular machineries to interpret the
information in these signals. Very different molecular features can influ-
ence protein sorting, and the distribution of a protein is dependent on a
cell's ability to decipher multiple signals operating at different stations
in the vacuolar system. Several excellent reviews cover much of this in-
formation (Bonifacino et al. 1996; Bonifacino and Traub 2003; Hirst and
Robinson 1998; Schmid 1997; Sorkin 2000; Trowbridge et al. 1993). Here
we give only superficial coverage of a few examples that are particularly
relevant to viral protein trafficking.

Aside from the sequences that specify ER translocation, protein fold-
ing and ER quality control (see the chapter by Maggioni and Braakman,
this volume), specific signals regulate protein export from the ER and
transport through the stations of the vacuolar pathway. These signals act
as markers either for transport or for retention. Signals for transport al-
low proteins to be incorporated into transport vesicles and move effi-
ciently from one compartment to another. These signals often operate
by binding the proteins in which they are located to coat proteins that
form the vesicle, or by ensuring proteins are located in the membrane
domains that are incorporated into vesicles. Retention signals operate in
the opposite way. They prevent incorporation of proteins into transport
vesicles and cause them to be retained in a specific membrane system.
These signals may operate by linking membrane proteins to cytoskeletal
elements, or keeping proteins in membrane domains that do not form
vesicles. Note that the well-characterized C-terminal KKxx (where x =
any amino acid) retrieval signal, that prevents export of ER proteins, is
in fact a transport signal that specifies incorporation of proteins into
retrograde transport vesicles operating between the Golgi and the ER
(Letourneur et al. 1994). Many proteins contain multiple signals, several
of which may allow efficient transport between several compartments
while others restrict transport once the protein has been delivered to a
specific target destination. Proteins that lack signals are transported in-
efficiently between compartments through non-specific inclusion in
transport vesicles.

The molecular features of sorting signals are varied and can include:

1. Short linear amino acid sequences. Best exemplified by the tyrosine-
based signals that mediate endocytosis. Two types of Y-based motif have
been identified. Yxx0 type signals (where = a large hydrophobic ami-
no acid) are found in the cytoplasmic domains (at least 7 residues from
the transmembrane domain) of many cell surface receptors and other

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R. Byland • M. Marsh

proteins that undergo endocytosis in clathrin- coated vesicles (CCVs).
These motifs interact with the ju2 subunit of the CCV AP2 adaptor com-
plex (see below). A second Y-based motif, FxNPxY, is found in the low-
density lipoprotein receptor and some other proteins that also undergo
endocytosis in CCVs. However, other adaptors such as ARH or Numb
may be involved in recruiting these proteins into CCVs (Aridor and
Traub 2002; He et al. 2002). In addition to mediating sorting at the plas-
ma membrane, Y-based motifs can also operate at the TGN and may in-
fluence sorting in polarized cells, delivery to endosomes and other spe-
cific transport steps. Motifs such as di-leucine sequences can also medi-
ate endocytosis and TGN sorting. Motifs such as DxE appear to be re-
quired for efficient export of proteins from the ER (for detailed list of
ER export signals, see Bonifacino and Glick 2004). Others, such as acidic
clusters with a pair of adjacent serines that can be phosphorylated by ca-
sein kinase II (CKII) (Molloy et al. 1994; Schafer et al. 1995; Takahashi et
al. 1995), can specify other sorting steps (Aridor and Traub 2002). Ex-
amples of motifs involved in sorting are listed in Table 1.

2. Transmembrane domains. The number of amino acids in the transmem-
brane domain of a single-pass integral membrane protein can influence
its association with membrane sub-domains of different lipid composi-
tion. Because of the ability of cholesterol to render the acyl chains of
sphingolipids more rigid, cholesterol-rich membrane domains are a few
A thicker. Proteins with transmembrane domains of approximately
22 aa show a propensity to associate with these cholesterol-rich do-
mains, whereas proteins with a transmembrane domain of around 18 aa
tend to be excluded (Bretscher and Munro 1993). Conserved residues in
the transmembrane domains of some membrane proteins have also
been linked to association with detergent-insoluble membrane domains
(DIMs) and apical sorting (Lin et al. 1998).

3. Lipid modifications. A number of cell surface proteins are linked to the
plasma membrane by glycophosphatidyl inositol (GPI) moieties that
are added to certain transmembrane proteins during their synthesis in
the ER. The GPI moiety replaces the transmembrane domain. Other
proteins are post-translationally modified by the addition of palmitic
acid to cysteine residues in the cytoplasmic domain or the transmem-
brane domain/cytoplasmic domain junction. Myristoylated proteins
can also be palmitoylated on cysteines close to the N-terminus. These
lipid modified proteins can all show some propensity to interact with
cholesterol-rich microdomains, which may influence their distribution
in the plasma membrane and other membrane systems.

Trafficking of Viral Membrane Proteins

225

I— I

u
O

•i-H

CD
CD

CD
P*

<-> .5 ^p

o a u

'ft °

a
p

+->

CD
U

C
bO

i-H

+->

cd cd

+-» +-»

<D CD

CD G 3

CO ^

Ph Ph
< <

i-H

p o

W co

•i-H
+->

u
O

+->

cd
Ph

PQ PQ
bO bJD

b O

& ON

cd 73 <N

« tS 73

55 d +-»

•iH J-H fl j

> a> w

a! ,13 (U

Q U ffi

• ^H

+->

• i-H

C/3
O

+->

sol

£K 00 00 Ph

> > > .

M N to >

K t> ffi oo

Ph 3

& CO
bO ^

a
o

■

+->

S3 P*
cd Ph

+->

XI
X

ON
ON

v,-, S C7N

)S ft ^

% 3 <^

O^ W H

+->

^ H u

cd 43
O

Ph Ch

u
u
cd

< < u ^<

CO
O

H-»

• i-H

CO
Ph

Ph
O

a>
+->

• i-H

^H
PW

«> 00

C^
C7N

m ^

ON ^

o\ cd

cd co

4-» <D

CD CD

• i-H

•i-H
+H

• i-H

+-•

+->

co
O

ON
ON

oo ft

ON tj

2 13

ON
ON

^— »

On
c^

^H-H

^^^^

•

•i-H

Q jz; ^

•i-H

^ CLi

& o

> 00 o

> ft +J

oJ w o

&0 "-< ^

CO Ph P

< < pq

O u

i-H

5h ^^
O CO

Ph CI

k^ »iH

p< 2

W Ph

PQ ^H

00 tHh

* mJ rn W N W ^

3 ^»»> S> ^o

u > > ^ P ^

ffi co t> t> ffi ffi

> >

ffi ffi >

^H Ph

g
cu

P
00

•i-H

i-H

CU
+->
CO

13 hP

u H

• i-H ^

U CO

< +

•i-H

CD O

W) J3
cd >>•

«? pq

hJ hP q q

226

R. Byland • M. Marsh

4. Glycosylation. Carbohydrates have been proposed to act as apical target-
ing signals for some secretory and membrane proteins (see Scheiffele et
al. 1995; Yeaman et al. 1997).

5. Oligomerization and cross-linking. Cross-linking or oligomerization of
membrane proteins can lead to a change of trafficking routes (see, for
example, Ukkonen et al. 1986).

Trafficking between membrane compartments is mediated by trans-
port vesicles, many of which are transiently coated with protein com-
plexes. Three distinct coats have been characterized to date. COP II
coats are associated with vesicles that bud from the ER en route to the
Golgi apparatus, COP I coats are associated with retrograde transport
vesicles from the Golgi to the ER (Robinson 1987), and CCVs are associ-
ated with endocytosis, transport from the TGN and some pathways from
endosomes. Additional coats have been implicated in other transport
events but remain to be characterized in detail (Seaman and Williams
2002).

The sorting information contained within membrane and other pro-
teins is interpreted by the cell's transport and sorting machineries. The
best understood of these machineries are the clathrin-associated adaptor
complexes, and in particular AP2, for which detailed structural informa-
tion now exists (Collins et al. 2002). AP adaptors, of which four have
been identified (API -4), are heterotetrameric complexes composed of
two large (a,(5,y or S), one medium ju (-50 kDa) and one small a
(-20 kDa) adaptin (Hirst and Robinson 1998). AP2 is associated with
endocytic CCVs (cf. API and AP3, which are associated with TGN and/
or endosomally-derived CCVs). The large adaptins contain a core do-
main and a so-called C-terminal ear domain connected to the core by a
flexible linker. The linker of the /?-subunits contains a 'clathrin box' se-
quence that binds the adaptor to the N-terminal j3 propeller domain of
clathrin (ter Haar et al. 1998). The ju2 subunit is responsible for binding
Yxx0 type signals. The hydrophobic side chains of the Tyr and Y+3 re-
sidues bind into hydrophobic pockets located in the ju2 j5 sheet C-termi-
nal domain. However, the binding site is only accessible after phosphor-
ylation of threonine 156 by adaptor-associated kinase 1 (AAK1), indicat-
ing that access is regulated (Conner and Schmid 2002; Ricotta et al.
2002). AAK1 activity is governed by clathrin (Conner et al. 2003; Jackson
et al. 2003) linking recruitment of Yxx0- containing cargo to clathrin as-
sembly at the plasma membrane. Di-leucine motifs with an upstream
acidic residue (e.g. ExxxLL), appear to bind the y- and al-subunits of
API rather than the ^-subunit (Janvier et al. 2003).

Trafficking of Viral Membrane Proteins 227

Similar modes of recognition, and perhaps regulation, may occur for
other clathrin adaptor complexes (Ghosh and Kornfeld 2003a). Whether
the same applies for other adaptors, including GGA proteins (for review,
see Bonifacino 2004), and coats that are now being identified remains to
be established (Aridor and Traub 2002; Ghosh and Kornfeld 2003b).
How other types of sorting signals that do not rely on short peptide se-
quences are interpreted also awaits additional study.

Traffic of Viral Envelope Proteins

Viruses, in particular enveloped viruses, have learned to exploit the cel-
lular trafficking pathways to facilitate their replication. In some viruses
the trafficking itineraries of the envelope proteins are relatively simple
and may lead to the proteins being exported from the ER and delivered
to the cell surface. By contrast, for other viruses more complex itiner-
aries involving multiple signals have been identified. Viral proteins have
adopted many of the sorting signals found in cellular proteins, and they
exploit the cellular sorting machineries. In some cases viruses encode
adaptors, for example HIV Nef, to couple proteins to trafficking ma-
chineries with which they do not normally interact, or interact in a dif-
ferent manner, thus sorting cellular proteins to different sites in infected
cells. Here we discuss several different families of viruses and the traf-
ficking properties of some of their membrane proteins. Relevant target-
ing motifs are outlined in Tables 1 and 2.

3.1
Retroviruses

Retroviruses encode a single envelope precursor protein (Env), which is
synthesized on the ER, undergoes oligomerization (usually to trimers)
and is then transported via the Golgi apparatus to the cell surface. En
route, each Env protein is proteolytically cleaved by furin or a furin-like
protease to form a heterodimer consisting of a so-called surface unit
(SU) and a transmembrane unit (TM). In the case of the human immu-
nodeficiency virus (HIV), and the related simian immunodeficiency vi-
rus (SIV), Env is synthesized as a 160-kDa precursor that is cleaved to
produce gpl20 (SU) and gp41 (TM) (see the chapter by Maggioni and
Braakman, this volume). Env incorporation into virions has largely been
considered to be a function of the cytoplasmic domain of TM and its in-

228

R. Byland • M. Marsh

Table 2. Targeting motifs

Retroviruses

HIV-1 Subtype B

SIV mac 239

HTLV-1

HTLV-2

FIV

RVRQOY

klrqlli^lfssppsyfqqthiqqdpalptregkerdggegggnsswpwqieyihflirqli

pvlltwlfsncrtllsrvyqilqpilqrlsatlqrirevlrteltylqygwsyfheavqavwr

satetlagawgdlwetlrrggrw1la1prr1rqgleltll

rhlpsrvrIphBBk-pessl

qalpqrlqnrhnq^^npetml

dcirncihkilg^Kampevegeeiqpqmelrrngrqcgmsekeee

Herpesviruses

HSV-1 gB

HSV-2 gB

VZVgB

ryvmrlqsnpmkalyplttkelknptnpdasgegeeggdfdeaklaearemtr^^Hvsa
mertehkakkkgtsaIII

.y.y.y.y.y

vsamertehkarkkgtsamsskvtnmvlrkrnkariiiiihnedeagdedel

.'.'.'.'.'.'.'.'.'.'

yryvlklktspmkalyplttkglkqlpegmdpfaekpnatdtpieeigdsqntepsvnsgfd

pdkfreaqemik^HHvsaaerqeskarkknktsaHtsrltglalrnrrgHBrtenvt

HCMV gB

HSV-1 gE

VZVgE

ytrqrrlctqplqnlfpylvsadgttvtsgstkdtslqappsyeesvynsgrkgpgppss

dastaappytneqa^^^alarldaeqraqqngtdsldgqtgtqdkgqkpnBdrlr

HRKNGlliilKiil^^H"'

''\\\\\\\\\\'A'A'A'A'A'A'A\'

ACMTCWRRRAWRjVKSRASGKGPTffeVADSELYADWH^^^HQVPWLAPPERPDS

- :■ :■ :■ :■ :■:■:' :■ :■ :■ :■ :* :■ :• :* ;• ;■ ;• ;■;■:■

•:■:■:■:■:■:■:■:■:■:■:■:■:■:■:■:■:■:■:■:■:■:•

KRMRVKAYRVDKSPYNQSMYYAGLPVDDFEDSESTDTEEEFGNAIGGSHGGSSYTVYIDK

"■*•*•*•*,'. ................ ...................... ............................................ .'.v.*.*.*.

.*.*.*.*.*.*.*.*.*.*.*.*.*.*.*.*.*.*.*.*.*.•.

VZV gH

HSV-1 gH

( w MLCGN)SRLRFJHHPLT

KVLRTSVPFFWRRE

Orthomyxovriuses

INFLUENZA HA

...MGVYQ 1LAIYATVAGSLSLAIMMAGISFWMCS NGSLQCRICI

Rhabdoviruses

VSV-G

RVGIYLCIKLKHTKlCRQlYTBiiiNRLGK

Poxviruses

Vaccinia B5R

cscdknndqIkfhkBp

teraction with Gag (Cosson 1996; Vincent et al. 1999), but the fact that
most retroviruses and retroviral Env proteins will form pseudotypes and
also incorporate cellular membrane proteins argues against high-fidelity
sorting during retroviral assembly. For the most part, retroviral Envs
have short cytoplasmic domains. HIV and SIV are unusual in that their

Trafficking of Viral Membrane Proteins 229

TM cytoplasmic domain varies from 150 to 200 amino acids for different
strains of virus.

HIV and SIV Env Trafficking. HIV Env trafficking has been studied with
respect to the formation of virions. Thus most models propose that Env
must be transported to the cell surface, where in the main HIV assembly
occurs. In many cell types HIV does bud from the plasma membrane,
but the degrees to which Env is incorporated into these particles is un-
clear. Early studies suggested that much of the newly synthesized HIV
Env exported from the ER is transported to lysosomes (Willey et al.
1988). Recent work has suggested a complex itinerary for Env and that
this trafficking activity is crucial for viral pathogenesis (Blot et al. 2003;
Fultzetal. 2001).

All HIV and SIV Envs contain a conserved Yxx0 type signal close
to the junction of the cytoplasmic and transmembrane domains (see
Table 2). This sequence functions as an endocytosis signal in HIV and
SIV Env and in Env cytoplasmic domain reporter constructs (Bowers et
al. 2000; LaBranche et al. 1995; Rowell et al. 1995; Wyss et al. 2001), and
as a basolateral sorting motif in polarized epithelial cells (LaBranche et
al. 1995; Lodge et al. 1997b; Owens et al. 1991; Rowell et al. 1995; Sauter
et al. 1996). SIV Envs, with short cytoplasmic domains, can sponta-
neously gain a Y/C mutation in the Yxx0 sequence when the virus is
maintained in tissue culture (LaBranche et al. 1995). This mutation de-
stroys the sorting information in the motif and the cell surface expres-
sion of Env is dramatically upregulated, arguing that endocytosis nor-
mally keeps cell surface Env levels low (LaBranche et al. 1995). When
SIV, containing an Env gene in which the same Tyr codon is deleted or
replaced by that of another amino acid, is used to infect non-human pri-
mates the animals become infected but do not develop AIDS unless the
mutation reverts (Fultz et al. 2001). Thus, though not required for
growth in tissue culture, the trafficking activity of the Yxx0 signal is re-
quired for pathogenesis in vivo. In addition, the bulk of the SIV cyto-
plasmic domain is not required for growth in tissue culture as the Env
gene frequently gains a mutation that places a premature stop codon
about 20 amino acids into the cytoplasmic domain of TM. However,
these truncations revert when the viruses are introduced into primate
hosts. The functional activities of the full-length cytoplasmic domain
are unclear as, for SIV at least, truncated Envs can still be incorporated
into virions. Thus the requirements for the full-length tail are intimately
tied into the biology of these viruses in vivo and may involve key roles
for trafficking.

230

R. Byland • M. Marsh

Full-length HIV and SIV Env, or Env cytoplasmic domain containing
reporter constructs, in which the membrane proximal Yxx0 signal is in-
activated by mutation of the Tyr retain some capacity for endocytosis
and AP2 binding and show only modest increases in cell surface levels
(Bowers et al. 2000; Wyss et al. 2001) but no longer show basolateral
sorting (Lodge et al. 1994, 1997b). This indicates (1) that additional en-
docytosis information exists downstream of the membrane proximal
signal and (2) that the membrane proximal signal is the only component
of Env responsible for polarized sorting. The nature of the additional
endocytic activity is obscure. In HIV Env, a C-terminal di-leucine has
been implicated in API binding and mutation of this motif in conjunc-
tion with mutation of the membrane proximal Tyr has additive effects in
increasing Env cell surface expression (Wyss et al. 2001). Mutation of
analogous sequences in SIV Env had no effect on cell surface expression
or endocytosis (Bowers et al. 2000). However, truncation of SIVmac Env
at residue 767 increased Env incorporation into virions even in the pres-
ence of an intact Yxx0 motif (Yuste and Desrosiers 2003). How this mu-
tation affects the trafficking of Env remains to be established.

In addition to the endocytosis and basolateral sorting information,
other sorting signals must exist in Env. A significant amount of HIV
Env, or an Env cytoplasmic domain reporter protein, is located in the
Golgi apparatus and /or the TGN (Berlioz-Torrent et al. 1999; Blot et al.
2003). Antibody feeding experiments show that at least some of this ma-
terial is internalized from the cell surface and that Env cycles between
the plasma membrane and the Golgi/TGN (Blot et al. 2003). EM observa-
tions of HIV-infected macrophages also show prominent labelling for
Env on viruses in late endosomes, where the majority of infectious virus
assembles in these cells (Pelchen-Matthews et al. 2003; Raposo et al.
2002). Thus HIV-Env trafficking may involve transit from the cell sur-
face to early and late endosomes, from where it is directed to the Golgi
apparatus or the TGN. From this site it is either returned to the cell sur-
face or perhaps cycles to endosomes. Little is known of how HIV Env
traffics to late endosomes, although the fact that the membrane proximal
Tyr is preceded by a Gly (GYxx0) as in a number of late endosomal/ly-
sosomal proteins may be relevant (Bonifacino and Traub 2003).

The cellular machinery responsible for the trafficking of Env has been
identified to some extent. The membrane proximal GYxx0 in HIV and
SIV Env can bind jul and the AP2 complex (Bowers et al. 2000; Wyss et
al. 2001). This signal may also bind API and AP3 (Bonifacino and Traub
2003; Ohno et al. 1997). Binding to API appears to be weaker than to
AP2, and its functional relevance (and that of AP3 binding) is unclear,

Trafficking of Viral Membrane Proteins 231

although a role in sorting Env from the Golgi to endosomes and/or recy-
cling internalized Env to the TGN is possible. The membrane proximal
Tyr is crucial for both AP2 and API binding. In addition, for AP2 bind-
ing, the G at Y-l, P at Y+2 and at Y+3 all influence endocytosis (Boge
et al. 1998; Bowers et al. 2000; Sauter et al. 1996). In this respect, the
HIV/SIV membrane proximal signal conforms to a consensus fi chain
binding motif (Bonifacino and DelP Angelica 1999). Other possible Y-
based motifs in HIV and SIV Envs have not been found to have a major
role in sorting or endocytosis, although they may bind adaptors or
adaptor components in vitro. TIP47 (tail interacting protein of 47 kDa),
a protein that plays a role in recycling from late endosomes to the TGN
and binds a di-aromatic motif in M6PRs (Diaz and Pfeffer 1998), has
also been implicated in sorting HIV and SIV Envs, where a similar motif
(Y 80 2 W 80 3 in HIV-Ihib) is conserved. This interaction has been suggested
to be crucial for Env incorporation into virions (Blot et al. 2003).

The key component that drives retrovirus assembly is Gag. Virus-like
particles (VLPs) can be formed in many cell types when Gag is ex-
pressed with or without other viral components. Gag has been reported
to be targeted directly to the plasma membrane and in some cases to as-
sociate with detergent-insoluble microdomains (Lindwasser and Resh
2001; Ono and Freed 2001; Suomalainen 2002). Although in many cell
systems HIV particles and VLPs bud from the plasma membrane, in
some cells, for example macrophages, the majority of particles assemble
on endocytic membranes (Pelchen-Matthews et al. 2003; Raposo et al.
2002). How exactly Gag is targeted to these membranes is unclear. One
possibility is that Gag may interact with AP2 and be carried from the
plasma membrane on endocytic vesicles (Batonick et al. 2003). Alterna-
tively Gag may be targeted directly to endosomal membranes. Perturba-
tion of these membranes by overexpressing a dominant-negative form of
Vps4, a AAA ATPase involved in ESCRT recycling (see below), can in-
crease Gag association with endosomal compartments (von Schwedler et
al. 2003) as can modulation of phosphatidyl inositol phosphate distribu-
tion (Ono and Freed 2003). The traffic of Env from the plasma mem-
brane through endosomal compartments and back to the plasma mem-
brane may then enable Env to be captured by Gag particles budding at
different cellular locations.

Other Retroviruses. The presence of endocytosis signals in the Env cyto-
plasmic domain is not uncommon in retroviruses. The human T cell leu-
kaemia virus (HTLV-I), a 5- retrovirus, has been studied in some detail.
HTLV-I Env is required for infectivity and cell-cell fusion but is only ex-

232

R. Byland • M. Marsh

pressed at low levels on the surfaces of infected cells and when Env is
expressed in the absence of other viral components (Delamarre et al.
1997; Derse et al. 2001; Jassal et al. 2001; Nagy et al. 1983). The cytoplas-
mic domain of Env appears to play important roles in virus transmission
as its truncation can increase cell-cell fusion activity (syncytium forma-
tion) and decrease cell-cell transmission of the virus (Kim et al. 2003;
Pique et al. 1993). In common with many other retroviruses, the cyto-
plasmic domain of HTLV-I Env is short (28 aa), but it contains Tyr resi-
dues at positions 476 and 479, both of which are found in a Yxx0 con-
text. Mutation of either, or both, Tyr residues leads to loss of basolateral
targeting when this Env is expressed in polarized cells (Lodge et al.
1997a). In the absence of other viral proteins, HTLV-I Env is located on
intracellular membranes close to the nucleus as well as at the plasma
membrane. Mutation of Y479, but not Y476, increases Env cell surface
expression and the fusogenic activity of transfected cells. The HTLV-I
Env cytoplasmic domain binds both API and AP2 in pull-down experi-
ments and interacts with the jul- and ^2-subunits in yeast two-hybrid as-
says. Significantly, the interaction with jul can be abolished by mutation
of Y479 only, whereas binding of jul is affected by mutations in either or
both tyrosines (Berlioz-Torrent et al. 1999).

Thus, as with HIV and SIV, a putative endocytosis signals appears to
limit HTLV-I Env cell surface expression, but in other respects the traf-
ficking information encoded in this protein is less complex than that of
HIV/SIV Envs. The transfer of HTLV-I from infected to uninfected cells
has suggested a role for a so-called Virological synapse' (Bangham
2003). In infected cells it appears that Gag is sorted to these regions of
cell-cell interaction. It remains to be seen whether this Gag is associated
with preassembled particles in membrane-bound compartments, or
whether viral particles bud into the 'synapse' or are transferred from
one cell to another by another mechanism, perhaps involving cell-cell
fusion (Igakura et al. 2003).

In addition to HIV/SIV and HTLV-I, Yxx0 sequences have been found
in the Env proteins of, for example, the avian a-retrovirus, Rous sarco-
ma virus (RSV) and Mason-Pfizer monkey virus (MPMV). In RSV Env
the signal is functionally silent but is activated in Env proteins that are
deficient for palmitoylation or have modified transmembrane domain
sequences (Ochsenbauer et al. 2000). When the signal is active, Env
exhibits rapid endocytosis through CCVs. Whether this signal can be
modulated in infected cells at different stages of the virus life cycle is un-
clear. Beta-retroviruses such as MPMV differ from other retroviruses in
that their capsid assembly occurs in the cytoplasm of infected cells be-

Trafficking of Viral Membrane Proteins 233

fore Gag membrane association. A motif has been identified in the
MPMV Gag that targets the newly synthesized protein to the area of the
cell around the microtubule organizing centre (MTOC), where core as-
sembly appears to occur (Sfakianos and Hunter 2003). Subsequently, as-
sembled cores traffic to the plasma membrane for envelopment, budding
and release. A Yxx0 type motif in MPMV Env is required for these
events and suggests that trafficking of Env through the recycling endo-
some compartment is required to bring the core particles to the plasma
membrane (Sfakianos et al. 2003). Whether the core particles interact
with Env in recycling endosomes and traffic to the cell surface on recy-
cling vesicles is unclear.

3.2
Herpesviruses

Herpesviruses express multiple envelope glycoproteins. Alpha-her-
pesviruses, such as herpes simplex virus (HSV), carry -12 envelope gly-
coproteins, whereas /^-herpesviruses, such as human cytomegalovirus
(HCMV), encode -60 putative glycoproteins, many of which may be car-
ried in the viral envelope. Some glycoproteins, for example gB, gH and
gL, are essential for viral entry and are thought to be conserved in all
herpesviruses. Others appear to be included to facilitate the infection
and replication of specific viruses. These may encode additional recep-
tor binding molecules, to broaden the range of susceptible cells, or pro-
teins that have been implicated in the signalling events occurring early
after fusion that prime cells for replicating the incoming virus (Kledal et
al. 1998).

Glycoprotein B (gB) is a type I integral membrane protein that is es-
sential for the attachment and fusion of herpesviruses with their host
cells (Spear and Longnecker 2003). The proteins form homodimers or
trimers in some herpesviruses and, in HCMV, are proteolytically cleaved
to generate two associated polypeptides. Immunofluorescence analysis
of the subcellular distribution of HSV gB shows low levels of surface ex-
pression and intracellular vesicular staining that co-labels for the early
endosome antigen, EEA1. This intracellular localization is linked to the
presence of a YxxL motif that is conserved in the C-terminal cytoplas-
mic domain of all sequenced gBs (see Table 2) (with the exception of
Epstein-Barr virus gB) (Heineman and Hall 2001; Nixdorf et al. 2000;
Radsak et al. 1996; Tirabassi and Enquist 1998; Tugizov et al. 1999). Re-
moval of the YxxL motif in HSV gB increases the expression of the pro-
tein at the cell surface (Fan et al. 2002). Other potential trafficking mo-

234

R. Byland • M. Marsh

tifs can also be identified, for example HSV gB has a second Yxx0 motif
closer to the transmembrane domain, as well as a di-leucine signal be-
tween the two Y-containing motifs. However, mutation of either one or
both of these motifs has no obvious effect on HSV gB distribution. In
another a-herpesvirus, varicella zoster virus (VZV), gB is primarily seen
in the TGN and to a lesser extent at the plasma membrane (Heineman
and Hall 2001; Heineman et al. 2000; Wang et al. 1998). As for HSV gB,
the cytoplasmic tail of VZV gB contains a di-leucine and two Yxx0 mo-
tifs (Heineman and Hall 2001). Mutational analysis indicated that the
membrane proximal Yxx0 motif is required for Golgi localization and
the distal Yxx0 motif for endocytosis. Mutations in the di-leucine se-
quence do not affect protein distribution (Heineman and Hall 2001).
Whether these subtle differences in gB trafficking contribute to the fact
that VZV-infected cells form syncytia is unclear.

HCMV gB undergoes endocytosis and recycling in fibroblasts and
epithelial cells. Internalization is believed to be clathrin dependent
(Tugizov et al. 1999), and the internalized protein is seen to co-localize
with y8-COP, Rab4, Rab5, Rabll, API, VIP-21 and TGN46, suggesting it
may traffic through early and recycling endosomes and the TGN (Jar vis
et al. 2002; Tugizov et al. 1998). The cytosolic domain of gB contains sev-
eral potential signals for sorting to endocytic vesicles, including tyro-
sine- and di-leucine-based motifs as well as a cluster of acidic residues
(Tugizov et al. 1999). Mutations in the acidic cluster impair internaliza-
tion and abolish recycling (Tugizov et al. 1999). In contrast to the a-her-
pesviruses, HCMV gB is transported to apical membranes in epithelial
cells independently of other viral glycoproteins, demonstrating that the
protein also contains autonomous information for vectorial sorting
(Tugizov et al. 1998). Apical transport is abolished by partial deletion of
the 20-aa transmembrane domain of gB, and stretches of large hydro-
phobic amino acids in this domain have been implicated in raft-depen-
dent gB sorting (Tugizov et al. 1998). In addition, a deletion of the acidic
cluster in the cytoplasmic domain leads to mis-sorting of the protein to
basolateral membranes in polarized cells (Tugizov et al. 1998). After the
deletion, the potential of the cytoplasmic domain Yxx0 and di-leucine
motifs as basolateral sorting determinants may become apparent. How-
ever, the cytoplasmic domain of gB might be involved in other functions
in certain cell types. In U373 cells, gB induces syncytium formation reg-
ulated by its cytoplasmic domain. It appears to be dependent on rapid
concentration of gB into endocytic vesicles at the plasma membrane
(Tugizov et al. 1994, 1995). Whether this indicates a need to generate re-
gions of concentrated gB for fusion complex formation is unclear. Endo-

Trafficking of Viral Membrane Proteins 235

cytic trafficking of gB may also play an important role in virus assembly.
The gB incorporated into virions appeared to have trafficked over the
plasma membrane (Radsak et al. 1996), and envelopment of the virus
has been proposed to happen in endocytic organelles (Fraile-Ramos et
al. 2002; Jarvis et al. 2002; Radsak et al. 1996). Thus endocytosis of gB
may be crucial for targeting the protein to sites of virus assembly.

Glycoprotein E (gE) is found in a number of herpesviruses. Together
with gl, gE forms a high-affinity receptor for immunoglobulin Fc do-
mains (Johnson and Feenstra 1987). In HSV-infected epithelial cells, the
gE-gl complex is found at adherens junctions on the lateral surface of
cells and is able to bind junction components to promote HSV spread
(Dingwell and Johnson 1998; Wisner et al. 2000). HSV lacking gE/gl, or
expressing a gE-gl complex lacking the cytoplasmic domains, is unable
to spread in epithelial cell cultures (Johnson et al. 2001; McMillan and
Johnson 2001; Wisner et al. 2000).

HSV gE cycles between the TGN and the cell surface. In the early stag-
es of infection, the bulk of the protein is in the TGN and only small
amounts are found at the plasma membrane (Alconada et al. 1999). Lat-
er in infection, the Golgi complex fragments and increased levels of
gE/gl are seen at cell junctions together with the TGN marker TGN46
(Alconada et al. 1998, 1999; McMillan and Johnson 2001). The cytoplas-
mic domains of gEs in herpesviruses have four regions of sequence sim-
ilarity. A Yxx0 motif and an acidic cluster are highly conserved. The
acidic cluster contains one or more potential CKII phosphorylation sites
(Alconada et al. 1996,, 1999; Zhu et al. 1996). A further Tyr- containing
tetrapeptide, and a conserved aromatic residue surrounded by hydro-
phobic amino acids, may also contribute to trafficking. Truncation of
the acidic cluster in HSV gE leads to its partial mislocalization to the
plasma membrane, but a role for phosphorylation has not yet been
found. Mutation of the Tyr in the Yxx0 motif also leads to partial mis-
sorting, and truncation of all four putative signals leads to exclusive sur-
face localization (Alconada et al. 1999; Jones et al. 1995; Takahashi et
al. 1995). The association of gE with gl also influences trafficking. In
epithelial cells infected with gl-negative HSV mutants, neither gE
nor TGN46 is found on lateral surfaces, suggesting that although gE pos-
sesses information necessary for internalization and recycling, transport
to cell junctions is dependent on information in gl (McMillan and John-
son 2001). Thus, although the intracellular accumulation of gE/gl may
be associated with virus assembly, transport of gE/gl to junctional do-
mains may facilitate cell-cell spread of the virus (McMillan and Johnson
2001).

236

R. Byland • M. Marsh

In VZV, gE and gl form a complex shortly after their synthesis
through interactions that require two conserved Cys-rich regions in the
gE ectodomain (Alconada et al. 1998; Yao et al. 1993). As with the HSV
homologues, VZV gE/gl accumulates in the TGN but also cycles over the
cell surface (Alconada et al. 1996, 1999; Zhu et al. 1995, 1996). Three po-
tential sorting signals similar to those found in HSV gE have been iden-
tified in the VZV gE cytoplasmic domain; two Tyr- containing tetrapep-
tides and an acidic cluster containing putative CKII phosphorylation
sites (Alconada et al. 1996; Zhu et al. 1996). The concerted action of all
three of these motifs is believed to be required for the steady-state local-
ization of gE to the TGN and for cycling between the TGN, the cell sur-
face and endosomes. Interaction with API at least is involved in this
trafficking (Olson and Grose 1997; Zhu et al. 1995, 1996). When VZV gl
is expressed without gE it is exclusively localized to the plasma mem-
brane, while gE alone is internalized. Expression of both proteins leads
to co-localization in the TGN, indicating that gl does not contain sorting
signals and that its distribution is determined by sorting information in
gE (Alconada et al. 1998). Examples of the distribution of one protein
being dependent on signals in an associated protein are not restricted to
herpesviruses. The bunyavirus envelope protein G2 is transported to the
plasma membrane when expressed alone but is retained in the Golgi
when expressed with Gl which contains a Golgi retention signal (Chen
et al. 1991; Matsuoka et al. 1991; Melin et al. 1995; Ronnholm 1992).
Similarly in rubella virus, El is targeted to the Golgi through its interac-
tion with E2 (Hobman et al. 1995).

Glycoprotein H (gH) is the third most abundant envelope protein in
VZV after gE and gB. It is required for membrane fusion and forms het-
erodimeric complexes with gL. Formation of this complex is essential
for maturation and cell surface expression of gH (Forghani et al. 1994;
Hutchinson et al. 1992; Montalvo and Grose 1986; Rodriguez et al. 1993).
gH is also efficiently internalized from the surface of VZV- infected cells
and localizes to the TGN. Co-expression of recombinant gH and its
chaperone gL showed that internalization is independent of other viral
glycoproteins, suggesting that gH contains its own sorting information
(Pasieka et al. 2003). The cytoplasmic domain of VZV gH is only 12-
14 aa long. Nevertheless, gH internalization is clathrin mediated and in-
volves a Yxx0 motif (Pasieka et al. 2003). All gH proteins of a-her-
pesviruses may be internalized in this way, except for HSV gH, where an
endocytosis signal appears to be missing. In HSV, gH can form com-
plexes with gB, which may provide the missing endocytosis information.
The cytoplasmic domains of ji- and y-herpesvirus gH proteins are only

Trafficking of Viral Membrane Proteins 237

6-10 amino acids long and may be too short to contain functional traf-
ficking information (Gompels et al. 1988; Pasieka et al. 2003). Little is
known about the trafficking of gH's chaperone gL.

Other Glycoproteins. In addition to the key proteins required for virus
entry, recent studies have suggested that other viral and cellular proteins
are incorporated into herpesviral membranes. The cytomegaloviruses
are particularly intriguing as these viruses encode a number of heptahe-
lical putative heterotrimeric G protein-coupled receptors (GPCRs).
HCMV encodes three, of which at least two (UL33 and US27) are incor-
porated into the viral envelope (Fraile-Ramos et al. 2002). The third pro-
tein (US28) has been demonstrated to bind a range of chemokines in-
cluding the unusual membrane-linked chemokine fractalkine (CX 3 CL1).
US28 is constitutively active (Kledal et al. 1998). It is also located pri-
marily in late endosome-associated vesicles in infected cells, together
with UL33 and US27, and may well be incorporated into virions budding
into these vesicles (Fraile-Ramos et al. 2001, 2002). US28 traffics over
the cell surface. Its internalization is clathrin dependent and appears to
require constitutive phosphorylation (Mokros et al. 2002), but, in con-
trast to many other GPCRs, it is not dependent on y8-arrestins (Fraile-
Ramos et al. 2003). These trafficking activities may be co-ordinated not
only to permit inclusion of US28 into viral membranes (and subsequent
signalling functions in the membrane of the target cells after fusion) but
also to allow US28 to scavenge chemokines from around infected cells,
thereby modulating the efficacy of immune responses to viral infection
(Beisser et al. 2002).

How exactly trafficking information is co-ordinated in herpesviruses
to allow the components of these complex viral membranes to be
brought together at the sites of viral budding remains to be established.
Proteins that may shepherd key viral membrane proteins to assembly
sites have been proposed, for example U6 in HSV (see Newcomb et al.
2003), but analogous proteins have yet to be identified in viruses such as
HCMV.

3.3
Orthomyxoviruses

Influenza A viruses are the prototype viruses of the orthomyxovirus
sub-family. These viruses contain two main surface glycoproteins,
haemagglutinin (HA) and neuraminidase (NA). HA has been widely
used as a model for studies of protein folding, protein quality control

238

R. Byland • M. Marsh

and membrane fusion (see chapters by Earp et al. and Salonen et al., this
volume). It is a non-covalently linked homotrimer. Each monomer has a
short 10-amino acid C-terminal cytoplasmic domain containing three
cysteines, which can be palmitoylated. By contrast, NA is a homote-
tramer of a type II membrane protein.

A primary site for influenza infection is the epithelium of the respira-
tory tract. In infected polarized epithelial cells, both HA and NA are ex-
pressed on the apical surface, where they are associated with lipid rafts
(Scheiffele et al. 1997). The transmembrane domain of HA and NA can
specify apical targeting through association with DIMs. Detailed muta-
tional analysis of the HA transmembrane domain indicates that al-
though partitioning into DIMs is required for apical sorting it is not suf-
ficient and that certain conserved residues within the transmembrane
domain may be required for interaction with the DIM-associated apical
sorting machinery (Lin et al. 1998). No internalization or recycling ac-
tivity has been detected for HA.

3.4

Rhabdoviruses

Vesicular stomatitis virus (VSV), the prototype rhabdovirus, encodes a
single type I integral envelope glycoprotein G. G has been used exten-
sively to study protein export from the ER and transit through the Golgi.
Truncation of the cytoplasmic domain leads to slower export without af-
fecting folding or trimerization of the protein, suggesting the pres-
ence of export signals (Doms et al. 1988). Mutation of a DxE motif (see
Table 1) does not affect transport, but mutation of a six-residue se-
quence (YTDIEM, position 19-24 in the cytoplasmic domain) inhibits
efficient export (Nishimura and Balch 1997).

In epithelial cells, G is targeted to basolateral surfaces, from where
the virus buds (Boulan and Pendergast 1980). G also localizes to coated
pits in these domains through which it is internalized (Matlin et al.
1983). A Yxx0 motif in the G cytoplasmic domain has been implicated
in basolateral targeting as mutation of the single tyrosine (Y501) and/
or 1504 (positions 19 and 22 in the cytoplasmic domain, respectively)
cause the protein to appear on both the apical and basolateral surfaces
(Thomas et al. 1993). In common with some other basolateral sorting
motifs, Y501 could not be replaced by any other amino acid but 1504
could be replaced by other hydrophobic amino acids (Thomas and Roth
1994). G also undergoes endocytosis, although the rate is slower than
that observed for other proteins using Y-based signals. The VSV Tyr

Trafficking of Viral Membrane Proteins 239

motif does not play a role in internalization and the nature of the endo-
cytosis signal remains unclear (Thomas et al. 1993).

3.5
Poxviruses

Vaccinia virus ( VV) is the prototype member of the poxvirus family. VV
particles can exist in several different forms. The intracellular mature
form (IMV) found in the cytoplasm is wrapped by two tightly apposed
membranes derived from the ERGIC (ER to Golgi Intermediate Com-
partment). IMVs can acquire two additional membranes from the TGN.
These particles can be transported to the plasma membrane, where they
are released by fusion of the outer membrane with the plasma mem-
brane to generate extracellular enveloped virions (EEV) (reviewed in
Smith et al. 2002). The membranes acquired from the TGN contain five
glycosylated and one non-glycosylated virally encoded membrane pro-
teins. Four of these are not essential for EEV formation, but two, F13L
(Blasco and Moss 1991) and B5R (Engelstad and Smith 1993; Wolffe et
al. 1993), are required for envelopment and high-level production of in-
fectious EEV (Blasco and Moss 1991). B5R, a 42-kDa glycoprotein, has
also been implicated in cell entry, because antibodies against its extra-
cellular domain can neutralize the virus (Galmiche et al. 1999). In infect-
ed cells both F13L and B5R concentrate in the membranes of the TGN
(Schmelz et al. 1994); F13L is a cytosolic peripheral protein that requires
acylation for membrane association when expressed alone. B5R is a type
I membrane protein (Isaacs et al. 1992) that is believed to contain its
own sorting information. These sorting signals reside in the transmem-
brane and/or cytoplasmic domains of the protein, as they are sufficient
to direct chimeric proteins to the Golgi and ensure their integration into
EEVs (Katz et al. 1997). Studies with chimeric proteins also indicate that
the B5R cytoplasmic domain prevents accumulation of the protein in the
plasma membrane, by either Golgi retention or endocytic retrieval
(Ward and Moss 2000). This domain displays two motifs that might reg-
ulate surface expression, a tyrosine at position 310 and a di-leucine sig-
nal at positions 315/316 (see Table 2). Mutation of either or both of these
motifs increases the levels of B5R on the plasma membrane. Antibody
uptake experiments demonstrate that B5R is cycling from the Golgi ap-
paratus to the plasma membrane and back with the stage of transit
through the TGN being the slowest part of the cycle. Mutations of Y310
or the di-leucine motif impaired retrograde transport, whereas a double
mutation abrogated it (Ward and Moss 2000). However, the exact contri-

240

R. Byland • M. Marsh

bution of each signal and the mechanism of transport remain to be fully
established.

Virus Assembly

Assembly of new viruses requires the temporally and spatially co-ordi-
nated co-localization of all the structural components required to form
an infectious virus. For most enveloped viruses, assembly is often seen
as a budding process in which the assembling virion progressively de-
forms the cellular membrane into a bud that eventually pinches off as a
free virus particle. For viruses with rapid replication cycles these events
often occur at the cell surface (Rowell et al. 1995), but for viruses that
establish long-term sustained infections assembly must be balanced to
ensure the survival of infected cells. In this case, a high cell surface ex-
pression of viral proteins is likely to attract the attention of the immune
system and alternative routes for assembly may be taken by the virus.

In many cases the localization of envelope proteins appears to deter-
mine the site of virus assembly. For other viruses the process is more
complex. For example, it has long been believed that HIV and other
retroviruses bud exclusively from the plasma membrane. However, HIV
Env is internalized efficiently from the surface, either requiring that Env
must be relocated during assembly or suggesting that not all assembly
occurs at the cell surface. It is possible that Gag might mask the internal-
ization motifs on Env, and allow Envs to accumulate at the cell surface,
but in some cells at least infectious HIV is assembled intracellularly
(Pelchen-Matthews et al. 2003).

It is becoming increasingly clear that cellular components are re-
quired to facilitate the assembly and release of many enveloped viruses.
One of the most extensively characterized of these machineries is the
eESCRT (Endosomal Sorting Complex Required for Transport) (Katz-
mann et al. 2002), a set of protein complexes involved in sorting mem-
brane proteins to lysosomes.

4.1

ESCRTing Virus Release

In contrast to the inwardly directed formation of membrane transport
vesicles, such as CCVs, which, regardless of the compartment on which
they are formed, are released into the cytoplasm of the cell, viruses are

Trafficking of Viral Membrane Proteins 241

released into the extracellular space or into the lumen of an organelle
(which is topologically outside the cell). The majority of studies of vesic-
ulation have involved inwardly driven processes, i.e. vesiculation into
the cytoplasm. Recently it has become apparent that cells have a highly
conserved machinery for outward vesiculation, i.e. vesiculation towards
the outside of the cell or lumen of intracellular compartments (see
Fig. 1) (Katzmann et al. 2002). This machinery, initially characterized
through the analysis of yeast vacuolar protein sorting (Vps) mutants is
responsible for the sorting of enzymes and substrates destined for lyso-
somes into membrane domains which form small vesicles in the lumina
of endosomes generating organelles termed multivesicular bodies
(MVBs). Once delivered to lysosomes, these internal membranes are
preferential targets for hydrolytic degradation (Katzmann et al. 2002). In
addition to sorting to lysosomes, other outward vesiculation events have
now been recognized and may involve the same machinery. One particu-
lar example is the generation of exosomes, small 50- to 90-nm-diameter
membrane vesicles formed in antigen-presenting cells (APC) and loaded
with a range of molecules including MHC class II-antigen complexes
(Stoorvogel et al. 2002). These vesicles can be released from APC and
are thought to play an as yet undefined role in immune responses. The
formation of MVBs requires ESCRT complexes (ESCRT 0-3) that are re-
cruited sequentially to endosomal membranes. These proteins select
cargo (frequently mono-ubiquitinated membrane proteins) and sort this
cargo into membrane domains that bud into the endosomal lumen. The
ESCRT machinery is recycled through the actions of a AAA- type ATPase,
Vps4 (Katzmann et al. 2002).

The topology of these events is identical to virion budding, and it has
emerged that the ESCRT machinery is also essential for the release of
HIV and a number of other retroviruses, as well as rhabdoviruses,
filoviruses (e.g. Ebola) and possibly also influenza viruses (see Marsh
and Thali 2003; reviewed in Pornillos et al. 2002). The molecular mecha-
nism through which the ESCRT machinery facilitates assembly is best
understood for retroviruses, including HIV. The Gag proteins of these
viruses contain sequences that are essential for late steps in the assem-
bly/budding process. These so-called late (L) domains are autonomous
signals that can be moved around within a Gag or matrix protein, can
function in trans or be swapped between viruses. Mutations in these
L-domains cause inhibition of virus release after the onset of assembly
but before scission of particles from the donor membrane. With HIV
L-domain mutants, budding profiles can be seen aligned along the plas-
ma membrane like a set of lollipops (Pornillos et al. 2002). In HIV the L

242

R. Byland • M. Marsh

domain sequences are located in the C-terminal p6 domain of Gag. A se-
quence PTAP has been identified as key for L-domain function (Freed
2002; Pornillos et al. 2002).

Searches for the interacting partners revealed that the PTAP motifs
bind the protein Tsg 101 (tumour susceptibility gene 101), a component
of ESCRT-1. Further analyses have shown that depletion of TsglOl and
ESCRT-1 from cells by RNA interference (RNAi) inhibits HIV release
and that virus release is blocked at the same stage of assembly as L-do-
main mutants. Thus the notion has developed that, through Gag, HIV re-
cruits the cellular machinery required for outward vesiculation. Interest-
ingly the PTAP sequence appears to mimic the sequence PSAP found in
the cellular protein Hrs. Hrs is a component of ESCRT-0 that usually re-
cruits ESCRT-1 to the membrane (Bache et al. 2003; Pornillos et al.
2003).

4.2

Other Late Domain Sequences

In addition to the PTAP motif, other p6 sequences may contribute to the
activities of the HIV L-domain. Recent studies have implicated a se-
quence, which lies downstream of the PTAP motif as having L-domain
activity. This sequence (YxxL) can recruit a protein called AIP1/ALIX,
which interacts with both TsglOl and the CHMP4 component of the ES-
CRT-3 complex. Thus HIV appears to have adopted a belt and braces
ability to recruit ESCRT-3 either through TsglOl or through AIP1/ALIX.
However, the significance of the later interaction is unclear, as mutations
in the PTAP motif, or TsglOl knock down, alone inhibit release of infec-
tious virus.

The non-primate lentivirus, equine infectious anaemia virus (EIAV),
appears to use a YxxL as its primary L-domain motif. As with HIV, the
motif interacts with AIP1/ALIX to recruit ESCRT-3. This sequence can
be exchanged with the HIV PTAP motif, indicating that although the
two motifs recruit different effectors they both operate through the same
pathway.

Late domain sequences have been found in other enveloped viruses.
In the murine leukaemia virus (MLV) the sequence PPPY is required for
budding. The PPPY motif does not bind TsglOl, but instead appears to
interact with a Nedd4-like ubiquitin E3 ligase BUL I (Yasuda et al. 2002).
As TsglOl also binds ubiquitinated proteins it has been speculated that
this might be an alternative means to recruit the ESCRT machinery, per-
haps by-passing ESCRT-1. Indeed, TsglOl knock down by RNAi does

Trafficking of Viral Membrane Proteins 243

not affect MLV release. Although a number of different viruses use dif-
ferent L-domain motifs and different means to recruit ESCRT compo-
nents, assembly of all of them is inhibited by dominant-negative Vps4.
Vps4 appears to be required to recycle membrane-bound ESCRT com-
plexes. In the presence of dominant-negative Vps4 the ESCRT machinery
becomes irreversibly associated with endosomal membranes and is no
longer available to facilitate virus release.

Conclusions

The trafficking of viral proteins has long been of interest to virologists
and cell biologists. Indeed, viral membrane proteins have been crucial
models used for dissecting key aspects of membrane trafficking ma-
chineries. More recent studies have indicated that many of these proteins
contain multiple trafficking signals that allow them to adopt complex
trafficking itineraries in the endocytic and exocytic pathways. The im-
portance of these signals in the biology of different viruses is being in-
creasingly recognized. One of the key observations to emerge is that not
only can specific signals affect the distribution of a viral protein in a cell
in tissue culture but they are also crucial for the pathogenesis of the vi-
rus in vivo. Indeed, detailed studies of the Tyr-based signals in SIV and
HIV Envs are shedding new light on the biology of these viruses. Much
more remains to be learned: It is likely that novel signals remain to be
discovered and that information on the trafficking of viral integral and
peripheral proteins will continue to emerge. Some of this information
may prove to be useful in deriving novel strategies to combat viral infec-
tion. Whether or not this is the case, it is nevertheless important to un-
derstand the trafficking itinerary of the proteins in order to develop a
more complete understanding of the cellular mechanisms that underlie
virus infection.

Acknowledgements R.B. is supported by grants from the Roche Research Foundation
and the NIH (AI-49784) and M.M. by the UK Medical Research Council. We thank
Annegret Pelchen-Matthews for critical reading and colleagues who have contributed
ideas and comments on the manuscript.

244

R. Byland • M. Marsh

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Subject Index

AAAATPase 231
actin 74,89

- cortex 72, 81

- cytoskeleton 94

- filament 71, 74, 94

- polymerisation 78
adaptor-associated kinase 1

(AAK1) 226
adeno-associated virus (AAV)
adenovirus 123

- capsid 91

- cell entry 90

ADP ribosylation factor 6 203
aggresome 71
alphavirus 141
amantadine 16
amphipatic a-helix 140
antigenic

- drift 188

- shift 188

- variation 188
antiviral drugs 16
API/2 225
arterivirus 162

118

baculovirus 94, 95
brome mosaic virus (BMV)
bunyavirus 128
butanedione monoxime 79

150

canine parvovirus (CPV)
caveolae 1 1
cell surface receptor 4
cellular trafficking 220, 222
cerulenin 158
chaperones 179
chemokine 237
chloroplast envelope 150

225

36, 39, 51

CHMP4 242
cholesterol 48, 224
clathrin 9, 222, 234
clathrin-coated vesicles (CCV)
CNX 182
comovirus 158
conformational change
COP
-I 226

- II 155, 226

- £-COP 234
coreceptor 6, 17
coronavirus 162

cowpea mosaic comovirus 158
coxsackie-adenovirus receptor

(CAR) 6
cross-linking 226
CRT 182

C-terminal helix 35
cytochalasin 78, 88
cytopathic vacuoles type I 141
cytoplasm 122
cytoplasmic

- tail 47

-transport 82,84, 117
cytoskeletal

- filament 81

- proteins 87
cytoskeleton 69, 70, 81, 95

- cooperation 80
cytosol 123

cytosolic transport 83, 85
cytotoxic T lymphocyte 201

DC-SIGN 204

detergent insoluble membrane domain

(DIM) 224
dextran 118

256

Subject Index

disulfide bonds 184
DMV 162
DNA retrovirus 119
Drosophila 77, 158
DTT 185
dynactin 76, 82
dynamitin 87, 89, 93
dynein 76, 82, 87, 89

ectodomain 32, 46, 47

EDEM 187

endocytic entry 2

endocytosis 7, 8, 13, 80, 90, 117, 207

endosomal marker 143

endosome 222, 224, 230

Env gene 229

envelope protein 227

Epstein-Barr virus (EBV) 11, 233

equine

- arteritis virus (EAV) 162

- infectious anaemia virus (EIAV) 242
ER 157, 221

ERAD 1 86
Erolp 184
ERp57 183
Er v2 1 84

Escherichia coli 120
ESCRT 221,231,240
extracellular

- enveloped virion (EEV) 239
-matrix 110

F protein 37
FHV 163

Flaviviridae 159
flock house virus 158
fluorescent tag 80
folding 178
fusion

- peptide 42, 45

- protein

- - class I 30, 33, 34, 52

- - class II 31, 32, 38, 52

G protein-coupled receptor

(GPCR) 237
Gag 220, 228

GGA protein 227

glycophosphatidyl inositol (GPI) 224

glycoprotein 26, 86, 237

-B 233

-E 235

-G 238

-H 236

- oligomer 27

glycosylation 182, 220, 221, 226

Golgi apparatus 157, 210, 222, 226, 239

GTP hydrolysis 114

GTPaseRan 113

GTP-tubulin 73

guanidine 157

guanylyltransferase 142

helix bundle formation 50
hepadnavirus 119
heparan sulfate 4
hepatitis

- B virus 119

- C virus 140, 159
herpes simplex virus (HSV)

233

82, 205,

- type 1 120

herpesvirus 5, 86, 87, 124, 205, 233

- P 233
heterodimer 33
hexon 124
HPV protein 210
human

- cytomegalovirus 233

- immunodeficiency virus (HIV) 121,
9, 88, 200, 201

- C-terminal helix 50

- entry 49

- - Env trafficking 229, 230

- envelope glycoprotein (Env)

- fusion 35

- papillomavirus 210

- T cell leukemia virus (HTLV-I) 231

importing 117
influenza

- hemagglutinin (HA) 26, 30, 33, 43,
52

- virus 5, 10, 72, 127, 237

26,34

Subject Index

257

integral membrane protein 139
intermediate filament protein 71
internal fusion peptide 44
Italian ringspot virus 161

jasplakinolide 78

Kaposi's sarcoma 206
kinesin 75, 87

- N-type 75
Kunj in virus 159

L domain 241
latrunculin 78, 88
lentivirus 121, 201
lipid

- modification 224

- raft 48, 222, 238

- stalk 40

low pH activation 28
lysosomal marker 143
lysosome 157

macropinocytosis 12
MAP kinase ERK 116
Mason-Pfizer monkey virus

(MPMV) 232
measles virus 6
membrane

- dynamics 40

- fusion 1 1
-proteins 27

- penetration 14, 16
methyltransferase 142
MHC

- class I 190, 207

- class II 190, 241
microfilament 71
microtubule 73, 90, 91

- transport 84
mitochondrial membrane 158
molecular crowding 179
MTOC 93

multivesicular body (MVB) 222
murine leukemia virus (MLV) 242

- Env glycoprotein 46
myosin 74, 89

-II 75

myristic acid 220

Nedd4-like ubiquitin ligase 242

Nef 202, 203, 227

negative-sense RNA virus 127

nepovirus 158

neurotropic virus 82

neutral pH 4, 29

nido virus 162

nocodazole 88

nodavirus 158

non-clathrin mediated endocytosis 10

nonstructural protein 139,142

N-proximal a helix 203

N-terminal

- fusion peptide 43

- myristoylation 201

- peptide 50

- zinc finger 208
NTPase 142
nuclear

- envelope 111

- export signal (NES) 113

- import 121, 126

- localization signal (NLS) 114

- pore complex (NPC) 111,114
nucleocapsid morphogenesis 95
nucleo-cytoplasmic transport 113
nucleoporin 112, 114, 115
nucleus 113, 122

oligomerization 226
orthomyxovirus 127, 237

palmitoylation 144
papilloma virus 125
papovavirus 125
paramyxovirus F protein 36, 37
parvovirus 15, 118

- entry 91
PDI 184

peptide translocation 205
PHD 208

phosphorylation 13
Picornaviridae 10, 14
PKC 14

258

Subject Index

plant virus 158
plasma membrane

124
polarized

- cell 222, 232

- epithelia

— infection 7

— receptor 7
poliovirus 140, 154, 164
polyoma virus 125
Polyomaviridae 1 1
poxvirus 92

protein sorting 221
proton pump 210
PTAP motif 242

Sindbis virus 141

8, 68, 72, 92, 93,

Rab protein 13, 234
rabies virus 77

Ran-independent transport 116
receptor 2, 6, 17

- activation 29

- cytoplasmic tail 207
redundancy 181
retention 186
retrotransposon 120
retrovirus 120, 227, 231

- alpha-retrovirus 29, 232

- budding 89

- ^-retrovirus 231
reverse transcription 123
rhabdovirus 238

RNA

- capping 142

- helicase 142

- polymerase sub unit 142

- replication 139

- triphosphatase 143

- virus 139

Rous sarcoma virus (RSV) 232
rubella virus 150

Semliki Forest virus (SFV) 140, 164

- fusion protein 38
signal peptidase 177
simian immunodeficiency virus

(SIV) 201

- Env trafficking 229

snurportin 117
sphingolipid 48
stalk model 40

TAP 205
targeting 176
TBE E 44
- homodimer

- protein
tegument protein 125
TGN46 234

TIP47 225, 231
tobacco

- etch potyvirus 158

- mosaic virus (TMV) 150
Togaviridae 150
Tombusvirus 161
tonoplast 150
trafficking 221

- signal 243

trans-Golgi network (TGN) 155, 221

translocation 114, 176

transmembrane domain 41, 45, 224

transposition 121

tubulin 74

tumour susceptibility gene 101 (TSG

101) 242
turnip yellow mosaic virus 150

ubiquitination 208, 209

UL33 237

unfolded protein response (UPR)

US27 237
US28 237

187

vaccinia virus (VV) 92, 239
vacuolar membrane ATPase 204
varicella zoster virus (VZV) 234
vesicular stomatitis virus (VSV) 238
VIP-21 234
viral

- capsid transport 86

- envelope 175

-genome, nuclear import 117

- protein trafficking 223

Subject Index

259

- subversion 189 - recombination 189
virion 70,88,126 virus -like particle (VLP) 231
virus Vps4 231

- assembly 240 Vpu 209

- entry 3, 16, 83

- receptor 4 Wiskott-Aldrich syndrome protein 94

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