Rho’ing in and out of cells

Abstract

Rho GTPases are key regulators of actin and microtubule dynamics and organization. Increasing evidence shows that many viruses have evolved diverse interactions with Rho GTPase signaling and manipulate them for their own benefit. In this review, we discuss how Rho GTPase signaling interferes with many steps in the viral replication cycle, especially entry, replication, and spread. Seen the diversity between viruses, it is not surprising that there is considerable variability in viral interactions with Rho GTPase signaling. However, several largely common effects on Rho GTPases and actin architecture and microtubule dynamics have been reported. For some of these processes, the molecular signaling and biological consequences are well documented while for others we just begin to understand them. A better knowledge and identification of common threads in the different viral interactions with Rho GTPase signaling and their ultimate consequences for virus and host may pave the way toward the development of new antiviral drugs that may target different viruses.

Keywords: :

• Rho GTPase
• actin
• egress
• entry
• gene expression
• immune system
• transformation
• virus

Introduction

Cellular rigidity and structure, as well as crucial cellular processes including phagocytosis, cellular movement or intracellular communication all depend on actin.^1-4 Two different forms can be distinguished in the cell: monomeric globular actin (G-actin) and polymeric filamentous actin (F-actin), the latter consisting of two parallel strands of actin monomers.

The actin cytoskeleton is mainly regulated by the Rho family of GTPases (Rho GTPases).⁵ These monomeric low molecular weight proteins constitute a distinct family with 22 mammalian members within the superfamily of Ras-related small GTPases, which are subdivided in the Rac subfamily (Rac1, Rac2, Rac3, and RhoG), Cdc42 subfamily (Cdc42, TC10, TCL, Chip, and Wrch-1), RhoA subfamily (RhoA, RhoB, and RhoC) and other Rho GTPases (RhoE/Rnd3, RhoH/TTF, Rif, RhoBTB1, RhoBTB2, Miro-1, Miro-2, RhoD, Rnd1, and Rnd2). Rho GTPases regulate abovementioned cellular processes by linking membrane receptors to the actin cytoskeleton, thereby explaining their fundamentality and omnipresence throughout eukaryotic cells.⁶ Because they regulate numerous different functions, Rho GTPases are strictly controlled in a spatiotemporal manner and can be viewed as molecular switches that cycle between an active GTP-bound and an inactive GDP-bound form. This switch is tightly regulated by three sets of proteins: guanine nucleotide-exchange factors (GEFs) that catalyze the activating exchange of GDP for GTP,⁷ GTP-ase activating proteins (GAPs) that stimulate the intrinsic GTPase activity to inactivate the switch⁸ and guanine nucleotide-dissociation inhibitors (GDIs), whose role appears to be to block spontaneous activation.⁹

In their active GTP-bound state, Rho GTPases act through a conformation-specific interaction with their target effector proteins. The best characterized members of the Rho GTPases are RhoA, Rac1, and Cdc42, which show high degrees of conservation over higher vertebrates. Activation of RhoA leads to formation of actin stress fibers and focal adhesion assembly, Rac1 typically regulates the formation of lamellipodia or membrane ruffles, while Cdc42 induces the formation of protrusive filopodia. Importantly, Rho GTPase signaling not only affects the actin cytoskeleton but also stability, dynamics, and polarization of the microtubule network.¹⁰

Over the past decades, many of the molecular details of the signal transduction pathways that connect Rho GTPase activity to rearrangements of the actin cytoskeleton have been unraveled. Nevertheless, identification of downstream targets of Rho GTPases has proven to be challenging. A combination of affinity chromatography, protein purification and yeast-two hybrid screening revealed over 100 potential Rho GTPase targets.¹¹ RhoA, Rac1, and Cdc42 alone are capable of interacting with about 20–30 different proteins in a GTP-dependent manner, underscoring their broad signal transduction potential. Some of the main interaction partners of RhoA, Cdc42, and Rac1 are summarized in Figure 1.

Figure 1. Schematic overview of Rho GTPase signaling. Cdc42 and Rac1 signaling leads to activation of the common effector protein p21-activated kinase (PAK) and scaffold proteins belonging to the WASP/WAVE family, which are key regulators of actin nucleation and polymerization. Active PAK phosphorylates myosin light chain kinase (MLCK), thereby inactivating it and inhibiting myosin light chain (MLC) phosphorylation and contractility. PAK also phosphorylates and activates LIMK which may lead to phosphorylation of cofilin, thereby inhibiting its actin-severing function. Downstream targets of RhoA include the serine/threonine kinase ROCK which is mainly involved in the formation of stress fibers and focal adhesions. ROCK phosphorylates downstream MLC, leading to actin–myosin contractility. At the same time, ROCK inhibits MLC dephosphorylation by inhibiting MLC phosphatase (MLCP). LIMK is also a downstream effector of ROCK. The mammalian homolog of diaphanous (mDia) is another important RhoA effector mediating actin nucleation.

In brief, RhoA interacts directly with the formin mDia, which promotes the polymerization of actin into linear filaments.¹² RhoA also activates ROCK (Rho-associated kinase) which increases myosin light chain (MLC) phosphorylation directly and through inactivation of MLC phosphatase, resulting in activation of myosin II.¹³ LIMK is another downstream effector of ROCK, which phosphorylates and inhibits cofilin.¹⁴ Cofilin is an F-actin-severing protein. Mainly depending on the local availability of G-actin monomers, cofilin activity may lead to net actin depolymerization or increased actin polymerization and branching. Other RhoA effectors include members of the ezrin/radixin/moesin (ERM) proteins¹⁵.

Rac1 is thought to release WAVE from an inhibited complex, thereby activating the actin-polymerizing complex Arp2/3.^16,17 Active Arp2/3 mediates branching and elongation of existing actin filaments, generating a meshwork. Cdc42 also activates Arp2/3 through a direct interaction with the Arp2/3 activators Wiskott-Aldrich syndrome protein (WASP) or N-WASP.¹⁸ Both Rac1 and Cdc42 also activate group I serine/threonine p21 activating kinases (PAK). These different signalization branches are interconnected. In general, RhoA pathway signaling counteracts the Rac1 and Cdc42 signaling axes and vice versa.

Because Rho GTPase signaling is involved in a plethora of cellular processes, it is perhaps not surprising that several viral gene products have evolved to interfere with and modulate these signaling axes. Indeed, several viruses interfere with the actin cytoskeleton and Rho GTPase signaling during many steps of their replication cycle. During entry and egress, these interactions may allow or facilitate viral particle passage across the cortical actin barrier and to rearrange actin to conformations that promote virus infection and spread, while other viral processes, such as intracellular movement, gene expression and latency, but also interaction with the immune system or oncogenesis may also depend to some extent on Rho GTPase signaling and associated actin rearrangements. In this review, the current knowledge of the interactions of different viruses with Rho GTPase signaling will be discussed, with an emphasis on processes that are conserved or similar in different viruses.

Entry

A first step during viral infection of a host cell consists of attachment of the virus particle to the surface of a host cell. In some cases, the initial host factors to which the virus particle adheres serve as genuine entry receptors and in other cases entry receptors must still be recruited upon this initial attachment phase.

A wide variety of strategies of viral entry in host cells have been described. Irrespective of the entry route, at some point, the plasma membrane itself and the cortical actin barrier that is located just beneath the plasma membrane need to be overcome.^19,20 Many viruses make use of cellular internalization processes for transport across the cell membrane and the cortical actin layer (endocytic viruses). Some viruses, however, enter by fusing their envelope with the plasma membrane (fusogenic viruses).²¹ During the latter type of entry, the cortical actin barrier is often remodeled by viral proteins to allow access of the particle to the cytoplasm.

It may come as no surprise that Rho GTPase signaling is involved in the entry process of many viruses (Fig. 2) but the exact contribution of Rho GTPase signaling appears to depend largely on the entry route and the specific cell type. Indeed, Rho GTPase signaling is involved in the intrinsic signaling pathways that drive different endocytic uptake processes that may be (mis)used by viruses, particularly phagocytosis and macropinocytosis.^22-34 On the other hand, fusogenic uptake may not directly depend on Rho signaling, but, upon fusion, local actin rearrangements might be required to allow passage of the capsids through the cortical actin meshwork underneath the plasma membrane.

Figure 2. Overview of the role of Rho GTPase signaling in viral entry. Rho GTPases are involved in the different entry mechansims used by diverse viruses: membrane fusion, clathrin-mediated endocytosis, caveolin-dependent endocytosis, phagocytosis and other entry processes, and in viral transport to the nucleus. For each entry process, the involvement of Cdc42, Rac1 and/or RhoA is indicated, in green if the Rho GTPase is upregulated or activated, in red when the Rho GTPase is downregulated or inhibited. Abbreviations; Kaposi’s sarcoma-associated herpesvirus (KSHV), adenovirus 2 (AV2), human cytomegalovirus (HCMV), equine herpesvirus (EHV-1), human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), parainfluenza virus 3 (PIV-3), herpes simplex virus (HSV-1), pseudorabies virus (PRV), hepatitis c virus (HCV), simian virus 40 (SV40), avian reovirus (ARV), Japanese encephalitis virus (JEV), adeno-associated virus 2 (AAV2), vaccinia virus (VV).

Viral surfing and receptor clustering

Several viruses, including retroviruses, papillomaviruses, herpesviruses, poxviruses, dengue virus and vesicular stomatitis virus (VSV), engage Rho GTPase signaling even before entering the cell. In order to reach the cell body, virions attach to filopodia-like structures, leading to unidirectional movements toward the cell, followed by viral entry. This process, called viral surfing, was first described by Lehmann and colleagues for murine leukemia virus (MLV), avian leukosis virus, human immunodeficiency virus (HIV) and VSV in HEK-293-T cells³⁵ and relies on activation of myosin II and actin rearrangements (Fig. 4A).^35-37 Herpes simplex virus 1 (HSV-1) and dengue virus not only travel along filopodia during entry, but may also actively induce filopodia formation at this stage through activation of Cdc42 and Rac1 signaling.^38,39

Figure 4. Illustrations of viral use and manipulation of the cytoskeleton through Rho GTPase signaling during virus entry, egress, and interaction with immune cells. (A) Actin(green)-dependent surfing of Avian Leukosis Virus particles (red) along filopodia to the cell body during virus entry. (B-C) Rac1-mediated microtubule(green)-based transport of adenovirus particles (red) to the nucleus (blue) during virus entry. Cells in (C) were treated with the Rac1 inhibitor NSC23766 and show reduced association of virus particles with microtubules and reduced accumulation of virus particles at the nuclear rim. (D-E) Similar phenotype of actin-based cell projections induced by the alphaherpesvirus pseudorabies virus US3 protein kinase (D) and vaccinia virus F11 protein (E). (D) shows actin (red), microtubules (blue) and virus particles (green), (E) shows actin cytoskeleton. (F) Actin(red)-dependent expulsion of vaccinia virus particles (green) from infected cells during virus egress. (G-I) Effects of HIV Nef-PAK2 signaling on HIV interaction with immune cells. (G) Nef-mediated production of virus particle (white)-tipped filopodia (red) in dendritic cells. (H-I) HIV (H) is unable to disturb actin-dependent immunological synapse (red) formation between infected Jurkat T cells (green) and Raji B cells (blue), whereas wild-type HIV disturbs immunological synapse formation (I). Image sources: (A) Lehmann et al., 2005, J Cell Biol, (B,C) Warren and Cassimeris, 2007, Cell Mot Cytoskeleton, (D) Image courtesy of Herman Favoreel, (E) Valderrama et al., 2006, Science, (F) Cudmore et al., 1995, Nature, (G) Aggarwal et al., 2012, PLoS Path, (H, I) Rudolph et al., 2009, J Virol.

Rho GTPases also play a crucial role in another viral pre-entry process. Virion entry requires interactions with specific receptors and coreceptors that are not always readily accessible. Therefore, several viruses have evolved interactions that induce specific Rho GTPase signaling leading to transport of the virus particle to sites of viral uptake. Binding of the hepatitis C virus (HCV) E2 protein to its CD81 receptor on hepatocytes increases the levels of activated RhoA, Rac1, and Cdc42. This induces actin rearrangements that lead to lateral movement of the virus-receptor complex to the tight junctions, the site where its coreceptor claudin is expressed.⁴⁰ Internalization then occurs via a clathrin dependent endocytosis pathway.⁴¹

The cellular coxsackievirus and adenovirus receptor (CAR) is also located at tight junctions and therefore inaccessible. Group B coxsackieviruses first attach to the GPI-anchored protein decay-accelerating factor (DAF), which activates Abl kinase. This leads to Rac1-dependent actin rearrangements, permitting viral movement to the tight junctions, where virions are subsequently internalized via caveolin-associated vesicles.⁴²

Membrane fusion

Certain enveloped viruses, such as HSV-1, Sendai virus and many retroviruses, like the HIV-1, possess pH-independent fusion proteins, which allow direct fusion with the plasma membrane in particular cell types. Although direct fusion with the plasma membrane has been shown to lead to a productive infection for these viruses, this does not exclude the possibility that these viruses may also enter via endocytosis in the same and certainly in other cell types.

HIV infection is initiated by binding of gp120 to the CD4 receptor and to chemokine coreceptors like CXCR4, which results in membrane fusion. Binding of gp120 to CD4 initiates signaling to moesin, Arp2/3 and filamin A, resulting in actin-dependent entry receptor clustering.^43,44 This clustering initiates Rho GTPase signaling, which in turn results in actin rearrangements facilitating the movement of virions through the cortical actin meshwork. Both activation of RhoA and Rac1 are involved in these processes.^45-47 A possible explanation is that Rac1 is activated through the CXCR4 pathway while RhoA activation results from CD4 engagement. During this process, the activity of the actin severing protein cofilin is regulated in a bi-phasic manner. First, cofilin is phosphorylated and thus inactivated, possibly contributing to receptor clustering, followed by cofilin activation, which might contribute to loosening the cortical actin barrier and thus facilitating nuclear translocation.^48-50

Herpesviruses may also enter via fusion at the plasma membrane. The first report suggesting the involvement of Rho GTPases in alphaherpesvirus entry showed that HSV-1 entry in MDCKII cells was associated with activation of Cdc42 and Rac1, and that overexpression of genetically engineered Rho GTPases with altered activity influenced HSV-1 infectivity.⁵¹ This is in line with findings that pseudorabies virus (PRV), another alphaherpesvirus, induces Cdc42-dependent signaling upon infection in sensory neurons.⁵² For yet another alphaherpesvirus, equine herpesvirus 1 (EHV-1), entry into equine dermal (ED) or rabbit kidney (RK13) cells depends on activation of the RhoA effector ROCK, as inhibitors of this kinase or overexpression of a negative regulator of ROCK interfered with EHV-1 infection.⁵³ Attachment of the betaherpesvirus human cytomegalovirus (HCMV) to fibroblasts is associated with integrin- and EGF receptor-mediated signaling through Src and PI3K respectively to downregulate RhoA activity. This leads to a decreased phosphorylation of cofilin and the subsequent disruption of stress fibers.⁵⁴ However, other researchers have questioned a role for EGF receptor and associated signaling in HCMV entry in fibroblasts.⁵⁵

The respiratory syncytial virus (RSV) and parainfluenza virus type-3 (PIV-3) both belong to the paramyxoviruses. Their F glycoproteins mediate two different forms of fusion: fusion of the virus with the plasma membrane, enabling viral entry and fusion of the infected cell membrane with adjacent cell membranes, leading to syncytia formation. In both processes, RhoA involvement has been reported but the underlying mechanisms are still unclear.^56-59

In conclusion, Rho GTPase activation is a common mechanism involved in viral uptake, facilitating passage of the virus through the cortical actin layer and into the cytoplasm. Despite these apparent similarities, important differences exist, as there are different and sometimes contrasting contributions of the RhoA, Rac1, and Cdc42 signaling axes during these processes.

Endocytosis

Many mammalian viruses take advantage of different endocytosis mechanisms for their entry and infection.^23,26,61 Endocytosis is advantageous for the virus, as it delivers the virus particle/genome deep into the cytoplasm, bypassing the cortical actin barrier.⁶⁰

Endocytosis can be subdivided in clathrin-dependent and clathrin-independent endocytosis. Both types are hijacked by viruses and often involve Rho GTPase signaling.

Clathrin mediated endocytosis (CME)

During clathrin-mediated endocytosis (CME), actin-nucleation promoting factors steer the formation of an Arp2/3-dependent network of branched actin filaments where a clathrin-coated pit forms, providing forces for invagination.^24,25 Studies have reported that CME involves a large number of conserved proteins, whose order of action and sequential recruitment is of uttermost importance. The GTPase dynamin is critically involved in CME, as it is necessary for maturation of the clathrin-coated pit as well as pinching off the clathrin-coated vesicle.⁶¹

Adenovirus 2 entry through CME depends on actin assembly and is triggered by activation of PI3K and, subsequently, Rac1 and Cdc42. Peculiarly, PAK1, a central downstream target of Cdc42 and Rac1, is not activated during viral entry.⁶²

Kaposi’s sarcoma herpesvirus (KSHV) or human herpesvirus 8 have been reported to enter fibroblasts through CME.⁶³ The interaction of KSHV gB with its receptor α3β1integrin on these cells induces integrin signaling which results, via FAK, Src, and PI3K, in temporal Cdc42 activation followed by sustained RhoA and ezrin activation.^64,65 The importance of RhoA activation and Src phosphorylation for KSHV entry was later confirmed in overexpression experiments with dominant negative/active variants in HEK-293-T cells.⁶⁶

Rho GTPase signaling may not only affect clathrin-coated vesicle formation but also subsequent movement of vesicles. Particularly, during CME and other internalization processes, Src signaling mediates assembly of the plasma-membrane associated Ras activation complex, activating Rho and Rab GTPases, which not only is critical for the formation of various types of endocytic vesicles but also for their subsequent movement. The latter may play a role in intracellular movement of viruses, including KSHV, and will be discussed further on.^67-69

Clathrin-independent endocytosis

Clathrin-independent endocytosis (CIE) pathways cover a diversity of internalization routes, such as phagocytosis, macropinocytosis and caveolin dependent endocytosis (CDE) pathways. As actin dynamics play a role in many of these processes, Rho GTPase signaling is likely to play an important role in CIE of viruses.

Caveolin-dependent endocytosis

CDE occurs via caveolae, which are flask-shaped invaginations of the plasma membrane that belong to the cholesterol-rich microdomains (lipid rafts). In these regions, cholesterol molecules are intercalated between lipid chains, causing a local decrease of membrane fluidity. Caveolin-1 is the main structural protein of caveolae, essential for their formation and stability. Simian Virus 40 (SV40) entry through caveolae is well-documented, and studies on SV40 entry have contributed essential information to our understanding of CDE. SV40 transiently binds to caveolae leading to a transient breakdown of actin stress fibers and subsequent temporal dynamin and actin recruitment. This is followed by the formation of actin processes, contributing to intracytoplasmic transport of the viral particles.^70,71 During SV40 entry, integrin signaling activates the PI3K/AKT pathway leading to RhoA inactivation through the RhoGAP GRAF1. This results in the phosphorylation of ezrin and subsequent detachment of cortical actin from the plasma membrane, enabling subsequent viral uptake.⁷² Although it was initially thought that caveolae vesicles were delivered to an organelle dubbed the caveosome, more recent research has questioned the existence of such organelle.^73,74 Little is known about avian reovirus (ARV) cell entry, but infection has been reported to lead to p38 MAPK and Src mediated Rac1 activation in the early stage of the viral replication cycle, beneficial for caveolin1-mediated endocytic virus entry and productive infection.⁷⁵

Macropinocytosis

Over the past years, macropinocytosis has emerged as an uptake route used by a wide variety of viruses.³¹ Virus particles first activate signaling pathways that trigger actin-mediated membrane ruffling and blebbing. In the next stage, the ‘collapse’ of these cytoskeletal structures induces the formation of large vacuoles (macropinosomes) at the plasma membrane, resulting in internalization of virus particles and subsequent penetration into the cytosol. Not surprisingly, Rho GTPases play a central role in macropinocytosis. Although small variations on the theme are reported, the pathways inducing macropinocytosis are quite conserved, and involve Rac1 and Cdc42 activation that lead to actin ruffle and filopodia formation. PAK activation is indispensable during all stages of macropinocytosis. PAK activation contributes to the initial stages of actin remodeling and is later also involved in the macropinosome closure by activating C-terminal-binding protein-1/brefeldin A-ADP ribosylated substrate (CtBP-1 also known as BARS).^{31,32,37,76-79}

Macropinocytic uptake has been reported for many viruses including, among others, poxviruses, Ebola virus, influenza virus, adenoviruses, herpesviruses and retroviruses. For example, several labs have reported on the role of Rho GTPases and macropinocytosis during vaccinia virus (VV) entry^{27,37,77,78,80} For an extensive overview of the (subtle) differences in macropinocytic uptake between the different viruses we refer to a review by Mercer and Helenius.³¹ More recently, actin-dependent macropinocytosis-like uptake has been described for human papillomavirus type 16.⁸¹ Also recently, macropinocytic uptake has been suggested for two herpesviruses, the betaherpesvirus HCMV and the gammaherpesvirus KSHV. The viral gH/gL/UL128–131 complex of HCMV triggers integrin/Src/paxillin-signaling in monocytes, which promotes viral internalization through the regulation of dynamin and actin rearrangements, suggesting a macropinocytosis-like route of entry into target monocytes.⁸² KSHV binding to HMVEC-d cells induces ROS production and subsequent Rac1 activation, thereby amplifying the signaling pathways that are needed for macropinocytic uptake of this virus in this cell type.⁸³

Phagocytosis and other endocytic routes

For some herpesviruses, atypical phagocytosis-like endocytic uptake routes have been described, which are all clathrin- and caveolin-independent.

HSV-1 follows a phagocytosis-like uptake route in corneal fibroblasts (CF) and nectin1-overexpressing Chinese hamster ovary (CHO) cells, during which brief Cdc42 activation is followed by sustained RhoA activation.⁸⁴ Interestingly, as described higher, KSHV induces very similar Rho GTPase signaling in fibroblasts, but in this case, signaling culminates in clathrin-mediated endocytosis. Obviously, Rho GTPase signaling is critical for initiation of different internalization processes, but the particular endocytosis route activated appears to be determined by additional, cell type-dependent factors. In line with the HSV-1 results, EHV-1 entry into CHO-K1 cells occurs via an endocytic or phagocytic mechanism that depends on ROCK activation.⁵³

Atypical entry processes that involve Rho GTPase signaling have also been described for other viruses. For the picornavirus echovirus 1 (EV1), cell entry is initiated by integrin clustering, which induces PAK1 activation. This entry route is caveolin and clathrin independent but was reported to transport the virus to the caveosome.⁸⁵ However, since, as mentioned higher, the existence of the caveosome is highly questionable, the latter may be revisited. Adeno associated virus 2 (AAV2) entry occurs via clathrin-, caveolin-, and dynamin-independent endocytosis that depends on Cdc42 signaling.⁸⁶

For several viruses, involvement of Rho GTPase signaling in virus entry was reported, although the specific uptake routes are still unclear. For example, Japanese encephalitis virus (JEV),⁸⁷ poliovirus,⁸⁸ rotavirus,^89,90 and baculovirus⁹¹ all appear to depend on RhoA activation for their entry. Porcine circovirus 2 (PCV-2) infection also appears to rely on small Rho GTPase activity for infection, as inhibition with Clostridium difficile toxin B reduced PCV-2 infection in different cell types.⁹²

Movement to the nucleus

Many viruses that replicate in the nucleus rely on microtubule-based transport for long distance intracellular movement from the periphery to the nucleus, while actin filaments may play a role in short-range movements.^19,93-97 Rho GTPase signaling may also contribute to this viral transport to the nucleus, particularly through Rho GTPase-mediated stabilization (acetylation/detyrosination) of microtubules.

As already mentioned above, RhoA and Cdc42 signaling is induced upon interaction of the KSHV glycoprotein gB with integrins and subsequent activation of integrin dependent focal adhesion kinase (FAK).⁶⁴ Apart from facilitating entry, this activation also modulates microtubule organization, intracellular virus transport, and delivery of the viral genome to the nucleus.^65,98 Along the same lines, for HCMV and EHV-1, RhoA signaling and concomitant stress fiber breakdown during entry has been associated with efficient virus translocation to the nucleus.^54,99

Rho GTPase-mediated modulation of microtubules during entry is not confined to herpesviruses alone. In adenovirus infected fibroblasts, RhoA- and Rac1-dependent increased acetylation and detyrosination of microtubules has been observed, which is thought to enhance the ability of microtubules to “search and capture” incoming viral particles (Fig. 4B&C).^100,101

Likewise, in African Swine Fever Virus (ASFV) infection, Rac1-mediated microtubule acetylation is required for microtubule stabilization during viral transport.¹⁰²

Hence, several viruses make use of RhoA and/or Rac1 GTPase signaling to modulate microtubules for establishment of infection or nuclear delivery of the viral genome.

Gene Expression and Latency

Once the virus has entered the target cell and reached its compartment of replication, gene expression and subsequent production and assembly of new virus particles takes place. For several viruses, Rho GTPase signaling has been reported to influence gene expression.

Activation of RhoA by the guanine exchange factor p115-RhoGEF inhibits HIV-1 gene expression in T lymphocytes.¹⁰³ This has been associated with the ability of RhoA to inhibit transcriptional activity of the NFAT transcription factor, which may affect HIV gene expression through the NFAT-binding site in the HIV long-terminal repeat.¹⁰⁴ Conversely, Rac1-mediated signaling may enhance HIV gene expression. In support of this, CD28-mediated signaling via Vav and Rac1 contributes to HIV gene expression in T lymphocytes.¹⁰⁵ Rho GTPase signaling may also affect viral gene expression in a more indirect manner. For example, the influenza virus protein NS1 leads to decreased pRb phosphorylation through RhoA inhibition, which results in G0/G1 cell cycle arrest, thereby creating optimal conditions for influenza virus replication.¹⁰⁶

Also PAKs, central effector proteins in the Rac1/Cdc42 signaling axes appear to be involved in viral gene expression. Recently, PAKs were shown to physically interact with the viral transactivator Tax of the human T-lymphotropic virus type 1 (HTLV-1) which enhanced Tax’ ability to activate transcription of the HTLV-1 long-terminal repeats.¹⁰⁷ Quite contrary, PAK1 was reported to be involved in suppression of HCV replication. The mTOR downstream effector p70S6 kinase activates PAK1 and contributes to PI3K- and ERK-mediated inhibition of HCV RNA replication.¹⁰⁸

Viruses typically need full viral gene expression and a resulting productive viral replication at some point to spread to other hosts. However, some viruses also benefit from a stealth mechanism with very limited viral gene expression. Such a dormant infection, referred to as latency, allows them to persistently infect particular cell types in their host. Specific stimuli during the lifetime of the host may then trigger full viral gene expression and production and spread of virus. To favor latency, viruses have developed a number of strategies aimed at interfering with different aspects of the host cell signaling machinery, which may include Rho GTPase signaling, although the exact implication of Rho GTPase signaling in latency is currently poorly understood.

Herpesviruses are notorious examples of viruses that can cause latent infections, which typically ensures them of a lifelong infection of their host. In the case of gammaherpesviruses, latency occurs by maintenance of the viral genome in the nuclei of infected lymphocytes. M2, a protein encoded by murine gammaherpesvirus 68 (MHV-68), associates with the Rho exchange factor Vav, resulting in Vav hyperphosphorylation and Rac1 activation. In vivo studies showed that the interaction of M2 with Vav proteins is important for the establishment of a latent infection of MHV-68 in B cells.^109,110

Retroviruses like HIV also may establish latency. In a transcriptome study in cell lines latently infected with HIV, Cdc42 expression was found to be downregulated, which perhaps may be involved in cellular maintenance of latency.¹¹¹

Overall, although there is considerable evidence that Rho GTPase signaling may be involved in regulating viral gene expression during productive viral replication for different viruses, the potential contribution of Rho GTPase signaling during viral latency is less well documented thus far.

Virus Assembly and Egress

Many viruses also interfere with Rho GTPase signaling during later stages of infection, during virus particle assembly, egress, and/or spread (Fig. 3). In influenza virus infection, different mechanisms, all implying some kind of Rho GTPase signaling were reported to impact infectious virus production. Activation of Cdc42 increases transport of the viral envelope protein neuraminidase to the plasma membrane, the site of viral assembly, leading to increased virus production.¹¹² Furthermore, interaction of Cdc42 with the YRKL domain of the influenza virus matrix protein M1 is beneficial for influenza virus production, since it influences the function of M1 during budding of the virus.¹¹³ RhoA, on the other hand, is involved in downregulation of HDAC6 activity, leading to increased microtubule acetylation, which in turn is associated with enhanced virus assembly and release.¹¹⁴

Figure 3. Overview of the role of Rho GTPase signaling during late stages of infection. Rho GTPases are involved in late stages of infection for different viruses: projection formation, budding and release and exocytosis. For each process, the involvement of Cdc42, Rac1 and/or RhoA is indicated, in green if the Rho GTPase is upregulated or activated, in red when the Rho GTPase is downregulated or inhibited. Abbreviations: hepatitis c virus (HCV), Marek’s disease virus (MDV), human immunodeficiency virus (HIV-1), murid herpesvirus 68 (MHV-68), Epstein-Barr virus (EBV), vaccinia virus (VV), pseudorabies virus (PRV), herpes simplex virus 2 (HSV-2).

In recent years, PAK1 was reported to contribute to ERK phosphorylation and thereby enhance influenza virus production, although the mechanism involved remained elusive.^115,116 Another publication also reported ERK activation upon influenza infection, but showed that this was mediated by RhoA. Irrespective of the contributing triggers, further downstream, MLC phosphorylation appears to be at the crossroad of different signaling pathways induced by influenza infection, and is critically involved in virus replication. This might be explained by the fact that activated MLC mediates nuclear export of the ribonucleoprotein complex.^19,116

During later stages of infection, and especially during egress, viruses encounter similar barriers as during entry. Indeed, in order to exit the cell, newly formed virus particles again have to overcome the cortical actin and the plasma membrane.^19,20 In addition, during egress, viruses may manipulate the actin cytoskeleton to assist in viral spread to neighboring cells.

For herpesviruses, poxviruses and retroviruses these processes have been intensively studied and important steps in the signaling cascades driving these processes have been identified. For other viruses, Rho GTPase signaling has been associated with actin remodeling during later stages of infection, but the underlying mechanism or functional consequences remain unclear.

Over the past decade, it has become clear that lytic alphaherpesvirus infection results in alterations in Rho GTPase signaling that culminate in drastic rearrangements of the actin cytoskeleton. Common actin rearrangements include disassembly of actin stress fibers and/or the formation of cellular projections that are often branched and over 40 µm in length (Fig. 4D). Alphaherpesvirus-induced actin rearrangements have been associated with increased viral spread in cell cultures and could be attributed to the conserved viral US3 serine/threonine protein kinase for several alphaherpesviruses, including the porcine PRV, human HSV-2, bovine herpesviruses BHV1- and -5, and Marek’s disease virus (MDV) of poultry.^117-121 For PRV, it has been shown that virus particles migrate inside the US3-induced projections to neighboring cells, allowing intercellular virus transmission, even in the presence of virus-neutralizing antibodies¹²⁰.

A first suggestion that US3 may interfere with Rho GTPase signaling came from Murata and coworkers for HSV-2¹¹⁷, and has been unraveled in more detail for PRV US3. For PRV, it was shown that US3 leads to phosphorylation and activation of PAKs. Using PAK knockout cell lines, PAK1 was found to be involved in the formation of cell projections whereas PAK2 was crucial for actin stress fiber breakdown.¹²² In line with these findings, PAK signaling contributed to efficient cell-to-cell spread of PRV.¹²² Recently, Jacob and coworkers demonstrated that US3- and PAK-dependent cofilin activation plays a central role in the US3-induced actin rearrangements.^123,124 HSV-1 was also found to activate cofilin although this appeared restricted to the early stages of infection.¹²⁴ Interestingly, for HSV-1, US3 has recently been reported to stabilize microtubules, which further contributes to virus spread.¹²⁵

Actin rearrangements by the US3 protein kinase typically depend on the kinase activity of this protein.^126-129 However, an exception to this is the chicken alphaherpesvirus MDV. Intact and kinase-inactive US3 of MDV display equal abilities to induce actin rearrangements, although these are less dramatic compared with those of other alphaherpesviruses, as MDV US3 induces actin stress fiber disassembly but has not been reported to induce cell projections.¹³⁰ Another indication that interference of MDV with Rho GTPase signaling is different compared with other alphaherpesviruses comes from the finding that, unlike for PRV, the Rac1/PAK signaling axis appears inhibitory to MDV spread, whereas the RhoA/ROCK signaling axis promotes spread.¹³¹

Actin containing projections that contribute to viral spread have also been reported for the gammaherpesviruses Epstein Barr virus (EBV) and MHV-68. Since these viruses do not encode a US3 kinase ortholog, the underlying mechanism is different, and depends on a viral integral membrane protein complex consisting of BDLF2/BMRF2 in EBV and its ortholog gp48/ORF58 in MHV-68. Experiments with dominant active and negative RhoA constructs suggest that Rho GTPase signaling is critically involved in the projection formation, although further research should clarify their specific role since different studies found opposing roles for RhoA.^132,133

For poxviruses, particularly VV, the F11 protein has been reported to induce actin rearrangements that resemble those induced by alphaherpesviral US3. Indeed, comparable to US3 of alphaherpesviruses, F11 of VV is necessary and sufficient to induce actin stress fiber disassembly and formation of cellular projections (Fig. 4E). This has been associated with enhanced motility of infected cells and enhanced virus spread.^134,135 Furthermore, F11 induces a reorganization of cortical actin and increased microtubule dynamics, resulting in an increased tendency of microtubules to reach the cell periphery, thereby promoting access of the virus to the plasma membrane.^136,137 Although F11 was also shown to downregulate Cdc42 and Rac1 at 8hpi, only the ability of F11 to inhibit RhoA signaling to its downstream effectors appears to contribute to the effects on the actin and microtubule cytoskeleton.^135-138 Recently, F11 was shown to interact with the Rho-GAP myosin 9A via a PDZ-like domain. This is the first described example of a functional PDZ-domain in a viral protein and its interaction with myosin 9A is essential to downregulate RhoA activity.^136,139 Of particular interest, the ability of F11 to manipulate Rho GTPase signaling contributes to viral invasion in an in vivo intranasal infection model in mice.¹³⁴

F11-mediated inhibition of RhoA signaling is not the sole mechanism whereby VV interferes with actin regulating signaling pathways. Indeed, newly formed cytoplasmic VV particles use an interaction between the viral integral membrane protein A36 and kinesin to travel along microtubules to the cell periphery, where they fuse with the plasma membrane to form extracellular cell-associated enveloped virus (CEV).^140,141 These CEV sit on the cell surface and induce an outside-in signaling cascade initiated by the viral B5 protein to locally activate Src and Abl family kinases, resulting in tyrosine phosphorylation of the viral integral membrane protein A36.^142-148 A36 phosphorylation triggers the recruitment of a signaling complex consisting of Grb2, Nck, WASP-interacting protein (WIP), N-WASP and Cdc42, a process that was recently shown to also depend on recruitment of clathrin.^144,149-157 This signaling complex stimulates the actin-nucleating activity of the Arp2/3 complex, resulting in actin polymerization-driven propulsion of virus CEV particles to neighboring cells, thereby enhancing cell-to-cell spread of the virus (Fig. 4F).^{154,155,158-162} While extensive information is available about the viral and host factors that initiate actin polymerization, much less is known about the factors that contribute to subsequent virion release. The host phosphoinositide 5-phosphatase SHIP2 and the viral A34 protein were reported to act as gatekeepers to regulate virion release. SHP2 is recruited to the actin tails by Abl and Src family tyrosine kinases, N-WASP and the viral A34.¹⁶³

There are indications that this type of virus signaling and spread may be conserved over different poxviruses, like monkeypox virus and variola virus.¹⁶⁴ Similar actin-dependent propulsion of virus particles has also been seen upon infection with ASFV, but the underlying signaling pathways are still unknown, although they appear different from VV-induced signaling. Another important difference with VV is that the ASFV virus particles remain intracellular, whereas they are located extracellularly in VV.¹⁶⁵

Perturbance of Rho GTPase signaling and concomitant actin rearrangements have also been extensively documented in HIV infections. The HIV Nef (negative factor) protein is a key regulator of these processes. Nef is an important pathogenicity factor in the progression to AIDS. This multifunctional protein is involved in a variety of processes involved in HIV pathogenesis, including replication, immune evasion and spread. Nef interacts with and activates PAK2 in a fragile multiprotein complex that also contains Rac1, and the GEF Vav1. Several potential cellular substrates for the Nef-PAK2 complex have been reported. These include Bad,¹⁶⁶ Merlin,¹⁶⁷ cofilin,¹⁶⁸ and, as recently reported, also paxillin¹⁶⁹ and Mek1.¹⁷⁰ Nef has been implicated in changes in the actin cytoskeleton late in HIV-infection, consisting of the formation of long cellular extensions, which depend on the GEF activity of Vav.¹⁷¹ In podocytes, Nef induces disassembly of stress fibers and formation of lamellipodia via an interaction with diaphanous interacting protein (DIP), a regulator of RhoA and Rac1 signaling, which induces Vav and p190RhoGAP phosphorylation leading to Rac1 activation and RhoA inhibition.¹⁷² In line with this, Nef-expressing podocytes were recently shown to display enhanced Rac1 activity.¹⁷³ In infected dendritic cells, HIV Nef contributes, via diaphanous 2 (Diaph2), to the formation of virus particle-tipped filopodia that make rapid and multiple contacts with CD4 T cells, which may contribute to HIV spread (Fig. 4G).¹⁷⁴

Furthermore, the Nef-PAK2 interaction potently inhibits motility of fibroblasts through accumulation of phosphorylated, inactive cofilin, a process that is highly conserved among different nef alleles of HIV-1, HIV-2, and SIV.^{168,171,175,176} In addition, Nef has been reported to interact in a PAK2-dependent manner with components of the exocyst complex.¹⁷⁷ The exocyst complex is an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for the formation of nanotubes that interconnect cells. The authors therefore proposed that these interactions may be crucial for Nef’s ability to promote nanotube formation and enhance intercellular virus spread.¹⁷⁷

Besides Nef, the Tat protein of HIV also causes stress fiber disassembly, in addition to peripheral retraction and ruffle formation in human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells (HMVEC-L). This process occurs through PAK1 activation, but the biological consequences are not entirely clear.¹⁷⁸ Tat can be released from HIV-infected cells and extracellular Tat targets different types of uninfected cells, including endothelial cells.¹⁷⁹ The HIV-Tat/α_vβ₃ integrin interaction leads to activation of FAK, RhoA, NF-κB, and pp60src. This signaling cascade results in vivo in angiogenesis and possibly also in increased endothelial permeability, which may contribute to dissemination of HIV.¹⁸⁰

In addition to these well-documented interactions of HIV proteins with Rho GTPase signaling, more general reports indicate that Rho GTPase signaling is involved in HIV egress. Active Cdc42 is involved in HIV budding and release, whereas citron K, a RhoA effector, enhances HIV exocytosis.^181,182

Involvement of Rho GTPase signaling in late stages of infection for other viruses is much less documented. Rac1 is activated by the Dengue virus 2 E protein late in infection and may be involved in the interaction between actin and the viral E protein, although the biological consequences are not entirely clear.¹⁸³ For HCV, Rho GTPase signaling is involved in loss of polarity of hepatocytes upon infection. The HCV core protein downregulates Dlg1 and SHIP2, thereby inhibiting RhoA activity but activating Rac1, which disrupts the apobasal polarity.¹⁸⁴

Interactions with the Immune System

Rho GTPase signaling is also involved in the complex interplay between viruses and the immune system. Viral manipulation of Rho GTPase signaling may therefore also affect the ability of the host immune system to overcome a viral infection. As the HIV virus has been intensively studied and infects immune cells, most data in this part are based on HIV research. This section is subdivided in T-lymphocytes, dendritic cells, and monocytes/macrophages, according to the immune cells involved.

T lymphocytes

Since CD4+ T lymphocytes represent the main target cell population of the HIV virus, it is not surprising that the already described Nef-PAK2 interaction is involved in different aspects of the interplay between the virus and T-lymphocytes, affecting T-cell maturation and activation, T cell receptor (TCR) signaling and motility of T-lymphocytes, which all may contribute to the progressive loss of CD4⁺ T cells that leads to the acquired immunodeficiency syndrome (AIDS).¹⁸⁵

At the interface between a T-cell and an antigen-presenting cell or target cell, TCR engagement by MHC-presented antigens triggers actin rearrangements that control receptor clustering and the formation of the immunological synapse (IS) at the contact area between both cells. The IS is highly enriched in Src tyrosine kinases and other signaling molecules that are critical for T cell activation and IS formation depends on cytoskeletal structures, particularly actin dynamics. Evolutionary conserved alterations of IS formation and function by Nef-PAK2 include interference with cell spreading and actin polymerization upon TCR engagement via inhibition of N-WASP and a reduced recruitment of the Src kinase Lck to the IS (Fig. 4H&I).^186-188 Nef expression also results in endosomal Lck accumulation but this appears to be PAK independent.¹⁸⁷ At the same time, the nucleocapsid protein of HIV-1 promotes localization of virus structural proteins to uropods of polarized T cells, which may contribute to virus transfer via T-cell–T-cell contacts through so-called virological synapses, a process that depends on cytoskeletal remodeling.^189,190

Nef-PAK2 also affects T lymphocyte migration, as it leads to inactivation of cofilin, a downstream effector of PAK signaling, which results in reduced chemoattractant-induced T cell motility.^168,176 Importantly, the Nef-PAK2 interaction also interferes with T-lymphocyte circulation in vivo. Inhibition of lymph node homing, subendothelial migration, and cell polarization, but not diapedesis, were all found to depend on Nef’s ability to inhibit host cell actin remodeling. Nef-mediated interference with in vivo recirculation of T lymphocytes may compromise T-cell help and therefore may function as a HIV pathogenicity factor.¹⁹¹

In general, increasing evidence supports the hypothesis that the combined effects of Nef-PAK2 on T cell activity and signaling allow sufficient T cell activation necessary for optimal virus replication in this cell type, but at the same time prevent excessive antiviral T cell activation, ultimately leading to an increase in virus replication and spread. This strategy allows Nef to optimize viral replication in CD4+ T lymphocytes and at the same time disturb efficient antiviral immunity.¹⁹²

Measles virus (MV), a single-stranded, negative-sense, enveloped RNA virus also interferes with actin remodeling in T-cells. CD3/CD28-induced activation of the PI3K/AKT kinase pathway and proliferation is impaired in T cells upon contact with the measles virus glycoprotein (gp) complex. MV exposure results in an almost complete collapse of membrane protrusions in T cells, consistent with their inability to activate Rac1 and Cdc42 in response to TCR ligation. This is associated with reduced phosphorylation levels of cofilin and ERM proteins. Moreover, these events prevent CD3 from being efficiently clustered and redistributed to the central region of the immunological synapse. Thus, by inducing microvillar collapse and interfering with cytoskeletal remodeling, MV signaling disturbs the ability of T cells to adhere, spread, and cluster receptors essential for sustained T-cell activation.¹⁹³

Dendritic cells

Apart from its effect on T-lymphocytes, HIV also manipulates the dendritic cells (DC) arm of the immune response by triggering DC differentiation and migration. These processes may enhance HIV transmission, as virus can be transferred from DC to the main target cell, CD4+ T lymphocytes. Binding of HIV to DC, especially the interaction between the viral gp120 envelope protein and cellular receptors like DC-SIGN and CCR5, induces Rho GTPase signaling that may contribute to this. Although different types of signaling have been reported upon interaction between HIV and DC-SIGN, they generally appear to be associated with increased viral dissemination. For example, HIV interaction with DC-SIGN has been reported to lead to Rho GTPase signaling via the Rho-GEF LARG, which contributes to viral transfer to T lymphocytes via virus-induced DC-T cell synapses.¹⁹⁴ On the other hand, HIV interaction with DC-SIGN on immature dendritic cells has been reported to induce Src/Cdc42/PAK1/WASP signaling, resulting in the formation of membrane extensions that facilitate the transfer of virus particles from DC to CD4+ T-lymphocytes.¹⁹⁵ In addition, interaction between HIV-1 gp120 and CCR5 induces a similar signaling pathway through c-Src which results in the activation of several GTPases, including Cdc42. Activated Cdc42 induces complexing of Wasp, LSP1, and the Arp2/3 complex with β-actin, thereby enhancing podosome formation and migration of immature dendritic cells.^196,197 Also, through targeting of Vav and Rac1 signaling, recombinant Nef stimulates maturation of immature human DC and increased clustering with and activation of CD4+ T cells, which has been hypothesized to increase viral transmission.^198,199 On the other hand, in a murine DC line stably expressing Nef, the Nef-PAK2 connection was found to inhibit DC maturation and antigen presentation, which may impair development of antiviral immunity in vivo.²⁰⁰ Clearly, although the molecular players are diverse and may depend on particular cell types, maturation stages or circumstances, HIV manipulates Rho GTPase signaling in DC in several ways to increase virus transmission to its primary target cell, the CD4+ T lymphocyte and to avoid the development of a potent antiviral immune response.

Plasmacytoid dendritic cells (pDC) form a special subpopulation of DC that is of particular importance in viral infections, as these cells are major producers of type I interferon (IFN). Recently, IFN production upon recognition of HCV infected hepatoma cells by pDCs via CD81/CD9 tetraspanins was found to depend on Rac1 activation.²⁰¹ This is in line with findings that Rac1 signaling may play a central role in type I IFN production by pDC, as this signaling axis was found to lead to IKK-α phosphorylation and subsequent nuclear translocation of IFN regulatory factor 7 (IRF7), which is a critical event in type I IFN production.²⁰² IRF3 is a known determinant of a strong virus- and dsRNA-induced IFNβ response. For RNA viruses like the human H1N1 and avian H7N7 influenza A virus and Sendai virus, infection resulted in Rac1- and PAK1-dependent activation of IRF3, thereby inducing an antiviral interferon response.

Monocytes/macrophages

Also in monocytes and macrophages, expression of HIV Nef modulates Rho GTPase signaling and particularly leads to activation of the Rac1 signaling axis.

In monocytes, Nef-mediated Rac1 activation results in clathrin- and dynamin-independent endocytosis of immune costimulatory molecules CD80 and CD86, likely involving Rac1-based actin polymerization, which has been associated with viral immune evasion.^203,204 Also in monocytes, the HIV-1 matrix protein p17 binds the IL-8 receptor CXCR1. In this way, p17 mimicks IL-8, triggering rapid adhesion and chemotaxis of monocytes through a pathway that involves RhoA/ROCK.²⁰⁵

In macrophages, HIV Nef expression induces Vav-mediated, small GTPase–dependent cytoskeleton remodeling resulting in the formation of cellular projections or nanotubes. Such nanotubes could also be observed in macrophages in tissue from HIV-infected patients, and contacted B-cells. There is evidence indicating that nanotubes may shuttle Nef from macrophages to B cells, which impairs IgG2A and IgA class switching.²⁰⁶

Human rhinovirus also activates Rac1 signaling in macrophages, leading to p38 activation and release of the pro-inflammatory cytokine CCL2, which may contribute to the inflammatory microenvironment in the human airway upon infection with rhinovirus.²⁰⁷

Other interactions with the immune system

A recent report described a fascinating additional mechanism how the Nef-PAK2 signaling axis may influence the immune system and viral replication. In transfection studies, Nef-PAK2 was found to lead to secretion of ADAM proteases in so-called extracellular vesicles (EV), reminiscent of what had been described earlier for melanoma cells. EV formation occurs through the integrin-associated adaptor protein paxillin, and the ADAM proteases in the EV can cleave pro-TNFα to active TNFα. As a consequence, peripheral blood mononuclear cells (PBMCs) that ingested these EV released TNFα, which may promote viral replication by creating a favorable microenvironment.¹⁶⁹

Furthermore, HIV-mediated interference with Rho GTPase signaling in endothelial cells may affect brain homeostasis by disrupting the blood-brain barrier (BBB). The BBB prevents the entrance of circulating molecules and immune cells into the central nervous system by forming specialized brain endothelial cells that are connected by tight junctions (TJ). HIV infection of BBB endothelial cells induces activation of RhoA and its downstream effector ROCK. Activated ROCK phosphorylates the tight junction proteins occludin and claudin5 directly, resulting in diminished barrier tightness and enhanced monocyte migration across the BBB.^208,209 The Tat protein of HIV is of particular importance in this respect, as it may affect BBB integrity through several mechanisms, including decreasing occludin expression through activation of the RhoA/ROCK signaling pathway, altering the expression of ZO-1 (another tight junction protein) through induction of RhoA signaling and phosphorylation and activation of transcription factor cAMP responsive element binding protein (CREB) and translocating ZO-1 to the nucleus through Rac1 activation.^210,211 On the other hand, promotor activity and expression of the P-glycoprotein (P-gp), an ATP-dependent exporter essential for the proper function of the BBB, are upregulated upon Tat-mediated RhoA-expression.²¹⁰

Transformation

Several virus families are implicated in carcinogenesis.^212-215 Viruses that are associated with human cancers include hepatitis B virus (HBV) (liver cancer), papillomaviruses (cervical and other anogenital cancers), EBV (Burkitt lymphoma and nasopharyngeal carcinoma), KSHV (Kaposi sarcoma), and human T-cell lymphotropic virus (adult T-cell leukemia). Loss of cell polarity, increased motility and invasion are among the hallmarks of cancer progression and are associated with cytoskeletal rearrangements. It is therefore not surprising that for all oncoviral families, Rho GTPase signaling is involved to some extent in tumorigenesis.

HTLV-1 infection is associated with clonal expansion and transformation of mature T lymphocytes. The viral protein Tax plays a critical role in the pathogenesis of adult T cell leukemia. The modulation and deregulation of cellular signaling by Tax involves a range of pathways, including direct interaction with Rho GTPases.²¹⁶

HBV is the major cause of hepatocellular carcinoma (HCC). Several studies reported that the HBX protein enhances cell motility and metastasis by activating the Rho GTPases Rac1 and RhoA/ROCK.^217-219 Furthermore, HBX activates PAK1 and Src, thereby triggering Raf phosphorylation and translocation to the mitochondria. Mitochondrial localization of Raf protects cells from stress-induced apoptosis, thus promoting cell survival.^220,221 Recently, it was shown that RhoE is frequently downregulated in HBV-associated HCC, and reduced RhoE correlated with more aggressive HCC since RhoE suppresses the RhoA/ROCK axis, leading to reduced invasiveness.²²²

In tumor cells transformed by human papillomaviruses HPV-16 and HPV-18, the RhoG-specific GEF SGEF was reported to contribute to their invasive capacity. The viral oncoprotein E6 complexes with SGEF and the tumor suppressor human Discs Larg (hDlg), resulting in a strong enhancement of RhoG activity and increased invasive capacity.²²³

LMP-1, the major EBV transforming protein affects Rho GTPase signaling, leading to the formation of filopodia and actin stress fibers. Interestingly, both processes are regulated by different domains of the LMP-1 protein. Filopodia formation is triggered by Cdc42 through the transmembrane spanning region, while stress fiber formation is induced by RhoA activation through the CTAR1 domain.^224,225 This CTAR1 was shown to mediate PI3K/AKT activation which also contributes to the oncogenic properties of the virus, especially by promoting cell survival.²²⁴ For another oncogenic gammaherpesvirus, KSHV, the v-FLIP protein activates Rac1 by inducing COX-2 and its inflammatory metabolite PGE-2 to promote inflammation, angiogenesis and invasion.^226,227 Another KSHV protein, viral G-protein coupled receptor (vGPCR), contributes to cell transformation by inducing the Rac1-PAK signaling pathway.^222,228 Rac1 induction by vGPCR of KSHV infection also contributes to oncogenesis via the activation of ROS production, which promotes proliferation and angiogenesis.²²² Also in mouse mammary tumor virus infection, Rac1 and ROS were suggested to be involved in tumorigenesis.²²⁹

The small tumor (st) antigen of the polyomavirus SV40 is required for the transforming activity of SV40 in epithelial cells. Expression of the st antigen leads to upregulation of Rac1 and Cdc42 and downregulation of RhoA and concomitant deregulation of the actin cytoskeleton²³⁰ and st-mediated activation of the PI3K-AKT-Rac1 signaling pathway involved in transformation.²³¹

Similarly, the oncogenic envelope proteins of the betaretroviruses enzootic nasal tumor virus (ENTV) and Jaagsiekte sheep retrovirus (JSRV) and the oncolytic adenoviral E4ORF1 protein all depend on the PI3K-AKT-mTOR pathway to induce transformation.^232,233 For adenovirus, E4ORF1 induces AKT activation through Tiam1 and Rac1.²³³

Some viruses on the other hand are oncolytic by selectively replicating in and killing tumor cells. Interestingly, for the oncolytic Newcastle disease virus (NDV), Rac1 expression was reported to be sufficient to render non-tumorigenic cells susceptible to NDV replication which identified Rac1 as a potential oncogene.²³⁴

Concluding remarks and future perspectives

Rho GTPase signaling and corresponding actin dynamics may be implicated in almost every step of the viral replication cycle. Indeed, accumulating evidence shows that Rho GTPase signaling is involved to a greater or lesser extent in viral entry, gene expression, egress and spread, transformation, and interactions with the immune system. Furthermore, viral infection affects microtubule dynamics and organization through Rho GTPases, which may affect transport of viral particles in and between cells. RhoA, Cdc42, and Rac1 are the most important viral targeted Rho GTPases, but surely other currently un(der)investigated Rho GTPases are also important for viral infection. Crosstalk between these different proteins and associated signaling axes further complicates this field of research.

Viral genomes are compact with limited coding capacity and must rely on host gene products for efficient replication. This interaction of viral and cellular proteins has evolved such that cellular signaling pathways are modulated or bypassed in often fascinating ways. These interactions not only teach us how these signaling molecules participate in the viral replication cycle but also enhance our understanding of the processes in which they are naturally involved. A good example hereof is the use of the macropinocytic entry route by a growing group of viruses. Indeed, as it is becoming increasingly clear that many viruses use macropinocytosis as one of their entry routes, increasing information becomes available about the viral and cellular molecular players involved, which also provides new insights into macropinocytosis in general and the signaling pathways associated with it. Rho GTPase signaling, and more specifically PAK signaling, plays a central role in this context and it is fascinating to see that evolutionary very distinct viruses show substantial similarity in interfering with this signaling pathway to facilitate their entry into host cells.

Manipulation of Rho GTPase signaling is a common aspect of viral entry, although the specific Rho GTPase signaling axes involved are highly variable, depending on the entry route used by the virus. Importantly, evidence is accumulating that entry routes not only differ between different viruses but also between different target cells. The specific impact of the different Rho GTPases and their sometimes conflicting roles in entry processes merit further attention in the future. More insights into the different entry routes and why different mechanisms are used in specific situations and cell types might lead to the development of antiviral drugs that block viral entry in pathogenically important target cells. Interestingly, a RhoA-derived peptide was shown to possess antiviral activity against RSV, PIV-3, and HIV by interfering with syncytium formation and entry,^59,235 confirming the potential of targeting small Rho GTPases in antiviral strategies.

For several viruses, efficient intercellular spread depends on Rho GTPase signaling. Indeed, actin-based projection formation enabling the virus to reach and infect neighboring cells is well-documented for diverse viruses. Interestingly, the mechanisms leading to these structurally largely similar cell projections are quite diverse within and between virus families, highlighting the complexity of Rho GTPase signaling and indicating that different manipulations of these signaling networks may lead to similar morphological consequences. Eventually, Rho GTPase signaling often leads to the activation of common downstream proteins. It will therefore be important to obtain clear insights into the downstream targets of Rho signaling, as they may be valuable to identify putative common signaling nodes, potentially allowing the development of antiviral drugs that may interfere with several signaling cascades induced by a virus, or even be active against different virus families. In support of this notion, the identification of downstream effectors of Rho GTPase signaling revealed that MHC phosphorylation is at the crossroad of different signaling pathways induced by influenza A infection, making it a very interesting target in a strategy to reduce viral replication.^115,116 Such a strategy to attempt to identify common signaling nodes may be of particular importance in the context of transforming viruses. Since Rho GTPase signaling is involved in the tumorigenic properties of all major oncogenic viruses, it will be particularly interesting to investigate potential signaling similarities. The PI3K/AKT signaling pathway appears to be promising in this respect, and may open important avenues in the search for novel anticancer therapeutics.

Another important question that merits further attention in future research is timing and subcellular localization of specific Rho GTPase activation. Indeed, consequences of Rho GTPase signaling may vary greatly depending on localization, timing and duration of the signal. These considerations add an extra layer of complexity to this field of research. For many viruses, the involvement of Rho GTPase signaling during infection is still largely based on inhibitor studies, making it difficult to extract the exact contribution of these central signaling axes in virus biology.

An aspect of viral interactions with Rho GTPase signaling that appears to be underinvestigated thus far is its contribution to antiviral immunity. This has been quite extensively studied for HIV, with particular emphasis on the Nef-PAK2 signaling axis, but undoubtedly also plays an important role in the biology and pathogenesis of other viruses. A clear view on how Rho GTPase signaling contributes to antiviral immunity and, conversely, how viruses manipulate Rho GTPase signaling to mislead, tamper with or hide from the immune system will be of utmost importance to obtain a realistic view on the antiviral potential of drugs targeting Rho GTPase signaling.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Research of H.W.F. is supported by grants from the F.W.O.-Vlaanderen and the Special Research Fund of Ghent University. C.V.D.B. is supported by a post-doc grant of the F.W.O.-Vlaanderen. T. J. is supported by a PhD grant from the Agency for Innovation by Science and Technology in Flanders (IWT-Vlaanderen).