Actin and Rho GTPases in herpesvirus biology.

Abstract

Viruses have evolved a variety of interactions with host cells to create an optimal niche for viral replication, persistence and spread. The actin cytoskeleton of the host cell and actin-regulating Rho GTPase signaling pathways can be involved in several of these interactions. This review focuses on recent findings on herpesvirus interactions with actin and Rho GTPases during viral entry, replication in the nucleus and egress. Unraveling these often fascinating interactions might also provide additional insights into sometimes poorly known aspects of actin biology (e.g. its role in the nucleus) and in the development of novel antiviral therapies.

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Actin and Rho GTPases in herpesvirus
biology
Herman W. Favoreel1, L.W. Enquist2and Becket Feierbach2
1Department of Virology, Parasitology, and Immunology, Faculty of Veterinary Medicine, Ghent University,
9820 Merelbeke, Belgium
2Department of Molecular Biology, Princeton, NJ 08544, USA
Viruses have evolved a variety of interactions with host
cells to create an optimal niche for viral replication,
persistence and spread. The actin cytoskeleton of the
host cell and actin-regulating Rho GTPase signaling
pathways can be involved in several of these inter-
actions. This review focuses on recent findings on her-
pesvirus interactions with actin and Rho GTPases during
viral entry, replication in the nucleus and egress. Unra-
veling these often fascinating interactions might also
provide additional insights into sometimes poorly
known aspects of actin biology (e.g. its role in the
nucleus) and in the development of novel antiviral
therapies.
Actin filaments and actin-regulating Rho GTPase
signaling pathways
Filamentous actin is a key structure of the cytoskeleton in
every cell. The actin cytoskeleton is involved in many
crucial cellular processes, including providing cell integ-
rity, mobility and shape; driving cell division and contrac-
tion; and the uptake of extracellular molecules.
Actin filaments consist of polarized ATP-bound globular
actin monomers. These filamentous actin (F-actin) mol-
ecules can associate into bundles or networks through
actin cross-linking molecules. The best characterized
F-actin bundles and networks are filopodia, lamellipodia
and actin stress fibers. Formation of the different actin
bundles and networks is regulated by signal transduction
pathways that depend on small Rho GTPases [1]. Of the
22 identified mammalian Rho GTPases, the best charac-
terized are RhoA (stress fibers), Rac1 (lamellipodia) and
Cdc42 (filopodia). These Rho GTPases act as molecular
switches and alternate between their active GTP-bound
form and their inactive GDP-bound form. Transition from
the active to the inactive form is accompanied by phos-
phorylation and activation of different downstream effec-
tor molecules that initiate signal transduction pathways
and subsequent rearrangements in the actin cytoskeleton.
These three signal transduction pathways are interwoven
and the RhoA pathway generally counteracts the Rac1
and Cdc42 pathways [2]. Details of Rho GTPase signaling
networks can be found elsewhere [3].
Becauseofthemanycellularfunctionsinwhichtheyare
involved, it comes as no surprise that many intracellular
pathogens
signaling pathways. These interactions can occur at sev-
eral points in the life cycle of the pathogens, including,
although not limited to, internalization, intracellular
movement and intercellular spread [4,5].
There are recent indications that, in particular cell
types, herpesviruses also interact with actin and/or Rho
GTPases during three major phases of the replication cycle
of herpesviruses: entry into the cytoplasm, replication and
assembly in the nucleus and maturation and egress. This
review presents an overview of these data and points to
future directions in research in this new and potentially
important field of herpesvirus biology. Special attention is
given on the alphaherpesvirus subfamily of herpesviruses,
but relevant references to work on other herpesviruses are
also mentioned. Alphaherpesviruses are the largest sub-
family of the Herpesviridae and comprise many closely
related pathogens, including herpes simplex virus (HSV)
and varicella-zoster virus (VZV) in humans and several
animal pathogens, such as pseudorabies virus (PRV) in
pigs,bovineherpesvirus1incattle,equineherpesvirus1in
horses and Marek’s disease virus (MDV) in chicken.
interactwith actinand actin-regulating
Virus entry in host cells
Cell-type-dependent involvement of actin in herpesvirus
entry
Following uptake in host cells, herpesviruses travel to the
nucleus through microtubule-based transport (reviewed in
[6,7]). A direct role for actin in herpesvirus entry is less
obvious and seems to be cell-type dependent.
Alphaherpesviruses have been thought generally to
enter cells by direct fusion of the viral envelope with the
plasma membrane of a host cell. Such direct fusion occurs
in specific cell lines, such as Vero cells [8]. Inhibition of
actin polymerization in Vero cells has little or no effect on
virus entry, indicating that this type of entry does not
depend on actin directly [9,10]. However, accumulating
evidence suggests alternative pH-dependent and -indepen-
dent endocytic entry routes for alphaherpesviruses in a
variety of cell types, including primary human corneal
fibroblasts, human epidermal keratinocytes, C10 murine
melanoma cells, Chinese hamster ovary (CHO) cells and
HeLacells[11–14].Arecentreportindicatesaroleforactin
in one of these alternative entry pathways. Clement and
coworkers found that HSV-1 entry in primary human
corneal fibroblasts and nectin-1-overexpressing CHO cells
is associated with formation of actin-based filopodia and
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TRENDS in MicrobiologyVol.15 No.9
Corresponding author: Favoreel, H.W. (herman.favoreel@UGent.be).
Available online 30 August 2007.
www.sciencedirect.com 0966-842X/$ – see front matter ? 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2007.08.003

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subsequent uptake of the virus particles through a phago-
cytosis-like process [12]. In addition, cytochalasin D and
latrunculin B, two inhibitors of actin polymerization,
blockeduptakeofthevirusefficiently [12].Thedependence
of herpesviruses on actin polymerization for successful
entry might therefore rely on the entry route taken by
the virus in a particular cell type.
Rho GTPase signaling pathways during herpesvirus
entry
The viral gD envelope protein is essential for stable bind-
ing and for entry of nearly all alphaherpesviruses (except
VZV and MDV). Remarkably, this single protein interacts
with one of several cellular receptors, including nectin-1,
nectin-2, herpes virus entry mediator and modified
heparan sulfate [15]. Nectins are part of the immunoglo-
bulin receptor family and are of paramount importance in
gD-mediated herpesvirus entry into several cell types.
Theyconstitutecelladhesionmoleculesthatareassociated
with the actin cytoskeleton through a molecule called
afadin. In addition, they activate Cdc42 and Rac1, which
results in the induction of filopodia and lamellipodia in
epithelial cells and fibroblasts and in the formation of
synapses in neurons [16,17].
Using L cells expressing nectin variants unable to bind
afadin, Sakisaka and colleagues demonstrated that the
direct physical association of nectins with the actin cytos-
keleton is superfluous for herpesvirus entry [18]. However,
there is some evidence for nectin-mediated activation of
Rho GTPase signaling during herpesvirus entry, although
the biological significance is still largely unknown.
DeRegge and colleaguesreported that interaction of the
gD envelope protein with nectin-1 (and possibly other
nectins) during entry of the swine alphaherpesvirus
PRV in sensory neurons of the trigeminal ganglion, a
predominant site for herpesvirus latency, resulted in the
Cdc42-drivenformationofsynapticboutons [19](Figure1).
Similar gD- and nectin-dependent formation of synaptic
boutons has been reported for recombinant HSV-1 gD in
rat hippocampal neurons [17]. Synaptic boutons have been
described to represent sites of herpesvirus egress from
axons [19–21], suggesting a possible role for gD- and
nectin-induced synaptic boutons in intercellular viral
spread.
In support of Cdc42 signaling during alphaherpesvirus
entry, entry of HSV-1 into primary corneal fibroblasts,
Madin-Darby canine kidney II (MDCKII) cells and nec-
tin-1-overexpressing CHO cells results in a temporary
activation of Cdc42 [12,22]. In nectin-1-overexpressing
CHO cells, brief Cdc42 activation was accompanied with
a more extended RhoA activation and the induction of
filopodia-like structures within 30 min of virus attachment
[12] (Figure 1). Virus particles associated preferentially
with these filopodia, possibly facilitating ‘surfing’ on the
plasma membrane, an actin–myosin-driven transport pro-
cess that has been described to precede cellular entry of
different viruses [23]. The underlying reason why herpes-
viruses might induce filopodia-like structures on virus
binding to cellular receptors remains unclear. Although
speculative, it might be that these filopodia enhance sub-
sequent endocytic or phagocytic uptake of herpesvirus
particles [12] or that, by inducing these virus-capturing
structures on cells that display the appropriate virus
receptors, additional virus particles are recruited to
increase the chance of successful infection.
Hoppe and colleagues suggested a role for Cdc42 in
herpesvirus entry by over-expressing Cdc42 mutants in
MDCKII cells, finding decreased infectivity [22]. These
studies suggested that inhibition of viral infectivity
occurred after the virus had crossed the plasma membrane
and thus after anyvirus surfing had taken place (Figure 1).
Alphaherpesvirus entry can be associated with Cdc42-
driven processes, but there is not a consensus on the
activation of Rac1 and RhoA signaling during entry. There
is evidence for Rac1 activation but not RhoA activation
during HSV-1 entry in MDCKII cells and vice versa in
primary corneal fibroblasts and nectin-1 over-expressing
CHO cells [12,22]. One possible explanation for this differ-
ence in signaling might again be the mode of viral entry. In
CHO cells and primary corneal fibroblasts, Rho GTPase
signaling is associated with a newly described phagocyto-
sis- or micropinocytosis-like pH-dependent entry route of
HSV particles [12,24] (Figure 1). It is noteworthy that
phagocytic uptake of HSV is associated with activation
of RhoA and Cdc42 but not Rac1 [12]. Two common types
of phagocytosis have been described: complement receptor
3-mediated phagocytosis (associated with little membrane
protrusion and ruffling activity and reliant on Rho acti-
vation) and Fcg receptor-mediated phagocytosis (charac-
terized by extensive filopodia and lamellipodia formation
and requires Rac1 and Cdc42 activation) [25]. Although
speculative, this possibly suggests that a cell-type-specific
intermediate pathway is engaged during HSV entry.
Rho GTPase signaling might be a common feature of
herpesvirusentry because it also occursduring entry of the
gammaherpesvirus Kaposi’s sarcoma-associated herpes-
virus (HHV-8). The interaction between the HHV-8 gB
envelope protein and a3b1integrin receptors resulted in
induction of signaling through the RhoA, Rac1 and Cdc42
GTPases[26,27].ActivationofRhoGTPasesduringHHV-8
entry results in a profound reorganization of the actin
architecture but also results in increased acetylation
and stabilization of microtubules [27,28]. Interestingly,
this increased acetylation and stabilization, rather than
the alterations in the actin architecture, seems to facilitate
microtubule-mediated delivery of the capsids to the
nucleus [28].
Viral replication and capsid assembly in the nucleus
Thereisanemergingrolefornuclearactinintranscription,
chromatin remodeling, mRNA export and nuclear struc-
ture and integrity (reviewed in [29–31]). Likewise, nuclear
actin might have a role in viral infection. Herpesvirus
replication, gene expression and capsid formation take
place within the host nucleus. Live imaging of movements
of fluorescently labeled HSV-1 capsids in epithelial cell
nuclei demonstrated that capsids undergo directed move-
ments in the nucleus [32]. Capsid movement is both
temperatureandenergydependent.Usingtheactininhibi-
tor latrunculin A, Forest and colleagues concluded that
these movements were dependent on actin [32]. In
addition, the authors used the putative myosin inhibitor
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2,3-butanedione monoxime (BDM) to show that the move-
ments were myosin dependent. The authors speculated
that capsids could be using actin for movement, either
through actin-based polymerization or using the actin
filaments as tracks.
Herpesviruses induce the formation of actin filaments
in the nucleus
Using serial-section block-face scanning electron micro-
scopy (SBFSEM), which enables the acquisition of volume
electron-microscopic images, Feierbach et al. found that
PRV capsids are associated with filamentous structures in
the nuclei of infected neurons and epithelial cells [33].
Similar filaments associate with HSV-1 and HSV-2 nucleo-
capsids in infected central and peripheral neurons and
glial cell nuclei [34]. Such filaments might be a conserved
feature in alphaherpesvirus infections. Feierbach et al.
found that actin filaments are formed in the nucleus of
cells infected with PRV or HSV-1. Because these actin
filaments associated with viral capsids during capsid
assembly and were similar to the ones seen by SBFSEM
[33], the authors concluded that intranuclear filaments are
composed of actin. The function of nuclear actin during
infection is currently unknown. The formation of higher
order capsid assemblies is dependent on actin [32,33], but
the addition of actin-depolymerizing drugs does not affect
viral titers [35]. These seemingly conflicting results might
be owing to differences in cell type or might simply reflect
that higher order capsid structures are not essential for
efficient viral assembly. In any case, further research will
be necessary to elucidate the role (if any) of F-actin in
capsid assembly.
Do nuclear actin filaments assist in intranuclear trans-
port of herpes capsids (Figure 2)? Although a mechanism
for capsid movement in the nucleus is still unknown, PRV
capsids co-localize with the actin motor myosin Va in the
nucleus [33]. Class V myosins are among the most
thoroughly studied forms of unconventional myosins and
considerable evidence supports their role in the transport
of organelles and vesicles [36]. Myosin V is a two-headed
myosin that shows progressive movement along actin fila-
ments [36,37]. Myosin V is one of the fastest myosins,
moving along actin filaments at a rate of 300–400 nm s?1
[38], which is comparable with the rate reported for
directed movements of HSV-1 capsids [32]. Distinct from
how Listeria and vaccinia virus use the actin cytoskeleton,
herpesvirus capsids can move by myosin motors on nuclear
actin filament tracks rather than by using actin polymer-
ization-based movement (Figure 2).
What triggers the formation of nuclear actin filaments
during infection?
Recent data from baculovirus suggest that the trigger
might be a viral protein. Baculovirus-infected cells also
form nuclear actin filaments at the time of virus assembly;
these filaments co-localize with nucleocapsids and the
baculovirus major capsid protein binds to F-actin [39].
The baculovirus protein p78/83 shares homology with
WASP, a nucleator of actin, and promotes actin filament
polymerization directly [40]. Herpesviruses do not seem to
encode a homologous protein. Whether a herpesvirus
protein(s) nucleates actin filaments directly or actin fila-
mentformationisanindirectresponsetoinfectionremains
to be seen.
Figure 1. Involvement of actin and Rho GTPase signaling during herpesvirus entry. Herpesvirus particles associate with receptors on the cellular surface. This association is
followed by viral entry in the cytoplasm, either by direct fusion of the viral envelope with the plasma membrane lipid bilayer or through an endocytic route that can depend
on actin polymerization. Viral attachment and/or entry can be associated with activation of Rho GTPase signaling pathways that have been reported to lead to different
downstream effects, including formation of filopodia and formation of synaptic boutons. These events are cell-type specific.
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Actin is a component of the herpesvirus virion
Recently,Bainesandcolleagueshaveconductedanelegant
electron tomography study of HSV-1 undergoing envelop-
ment at the inner nuclear membrane (INM) [41]. These
images showed HSV-1 capsids connected to the INM by
fibers 8–19 nm in length and ?5 nm in width [41]. Fibers of
similar dimensions bridged the virion envelope and the
surface of the nucleocapsid in perinuclear virions. These
authors speculated that the fibers are composed of actin
owing to structural similarities between the fibers and
F-actin [41]. Recent work has shown that host-derived
actin is incorporated into the PRV virion and becomes
an integral part of the outer tegument layer [42,43]. Vir-
ion-associated actin has been reported in other herpes-
viruses [44–49] and other enveloped viruses, including
paramyxovirus, retrovirus and rhabdovirus [50–54]. But
why do herpesviruses contain F-actin in the virion? F-actin
could have a structural role or it might be a residual
product of an earlier role, such as facilitating the associ-
ation of the capsid with the INM (Figure 2). In any case, it
is unclear where within the host cell virions acquire actin
and if actin has any role in the virion.
Herpesvirus egress from the nucleus
Nuclear egress of alphaherpesvirus capsids involves
engagement with the INM and subsequent budding into
the perinuclear space through a tight meshwork of lamins
and associated proteins [55,56]. Infection alters the
nuclear structure through the reorganization of the lamin
proteins on the INM [35,57–59]. Simpson-Holley and col-
leagues have demonstrated that actin is required for
the characteristic nuclear expansion seen during HSV-1
infection of epithelial cells [35]. Emerin, a member of
the LAP-2, emerin, MAN-1 (LEM) domain class of INM
proteins, binds several nuclear components, including
lamins, F-actin and nuclear myosin I. Emerin also
enhances actin polymerization and might be involved in
stabilizing actin at the nuclear membrane, thus contribut-
ing to nuclear integrity (reviewed in [60]). Recently, Morris
and colleagues found that HSV-1 infection induces phos-
phorylation and delocalization of emerin, possibly result-
ing in changes in nuclear integrity [61]. Thus, herpesvirus
infection might disrupt nuclear integrity during egress by
the reorganization of INM proteins, possibly including
nuclear actin.
Virus envelopment and egress
Role of actin in late stages of alphaherpesvirus infection
As mentioned earlier, the physical connection of the her-
pesvirus entry receptor nectin with the actin cytoskeleton
seems redundant for herpesvirus entry [18]. However,
Sakisaka et al. found that it can facilitate viral spread
from one cell to another [18]. Because alphaherpesvirus
spreadinpolarizedcellsoccursthroughadherensjunctions
Figure 2. Herpesvirus infection induces the formation of actin filaments in the nucleus. Herpesvirus capsid assembly and genome packaging occurs in the nucleus in higher
order structures, which are dependent on F-actin for their formation. Following capsid assembly and packaging, capsids undergo movements towards the nuclear
envelope, possibly along actin filaments through the motor myosin V. On exit from the nucleus, the virus might acquire actin as an integral part of its tegument.
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[62] and because afadin binding is required for nectin-1 to
localize at cadherin-based adherens junctions [63], afadin
mighthavearoleinviralcell-to-cellspreadbydirectingthe
gD receptor to the required cellular site in specific cell
types. However, a role for the actin-binding properties
of nectin during alphaherpesvirus spread seems to be
cell-type dependent [64].
For PRV and MDV, for several cell types during
infection, expression of the viral US3 serine/threonine
kinase results in disassembly of actin stress fibers in
different cell types [65,66] (Figure 3). The US3 protein is
conserved among alphaherpesviruses and, although not
essential for alphaherpesvirus replication, is implicated in
several aspects of alphaherpesvirus infection, such as
egress of progeny virus from the nucleus and protection
of cells from apoptotic cell death [66–70]. PRV and
MDV US3 are necessary and sufficient to induce stress
fiber disassembly [65,66]. Transfection of HSV-2 US3 in
HEp-2 cells also results in a loss of actin stress fibers, with
a concomitant rounding of the cells [71]. PRV US3-induced
disassembly of stress fibers was also associated with cell
rounding and, in addition, with the induction of long, often
branched actin-containing cell projections in sparsely
seeded ST, RK13 and PK15 cells [72,73] (Figure 3). These
cellprojectionsestablishedintimatecontactswithadjacent
cells and virus particles migrated in the projections
towardsthetip.InhibitionofUS3-inducedactinrearrange-
ments using the actin-stabilizing drug jasplakinolide
reducedantibody-resistant
PRV significantly, suggesting a role for this process in
intercellularspreadof
enhancing intercellular virus spread [73]. Although
further experiments are needed to evaluate whether this
mode of virus spread might occur under natural circum-
stances of infection, it is interesting that similar virus-
induced cell projections have also been reported for HSV-1
in Vero and BHK cells and for VZV in HFF cells. It remains
to be determined whether the US3 orthologs of these
viruses are involved [74,75]. In addition, it is of interest
that, similar to US3 of other alphaherpesviruses, the
betaherpesvirus HCMV has also been shown to induce
actin stress fiber disassembly, through the UL37 exon 1
protein, a process that has been reported to occur by Ca2+
release from the endoplasmic reticulum and/or alterations
in mitochondrial bioenergetics [76–78].
Interaction of alphaherpesviruses with Rho GTPase
signaling during late stages of infection
The exact mechanism of actin rearrangements by US3
and possibly other viral proteins remains largely unstu-
died, although it is likely that Rho GTPase signaling is
involved. In support of this, co-expression of US3 with
dominant active forms of Cdc42 and Rac1 inhibited
HSV-2 US3-induced cell rounding and inhibition of
RhoA signaling mimics the effects of US3 in cells infected
with US3null PRV [71,73]. Our own data suggest that
the kinase activity of PRV US3 is required for its ability
to alter the actin cytoskeleton. It might therefore be that
US3 phosphorylates and thereby (in)activates one or
several components in the Rho GTPase signaling path-
ways.
Figure 3. Involvement of actin and Rho GTPase signaling during herpesvirus egress. Actin incorporation in virus particles might occur at late stages of infection, during
budding of cytoplasmic capsids in vesicles derived from the trans-Golgi network. Later stages in the herpesvirus replication cycle are also associated with disassembly of
actin stress fibers, rounding and retracting of cells and formation of actin-containing cell projections. These cell projections can be associated with enhanced viral
intercellular spread. Some of these events can be cell-type specific.
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