Herpesviruses Exploit Several Host Compartments forEnvelopment

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© 2012 John Wiley & Sons A/S doi:10.1111/j.1600-0854.2012.01399.x

Review

Herpesviruses Exploit Several Host Compartments for Envelopment Daniel Henaff, Kerstin Radtke and Roger Lippe´ ∗ Department of Pathology and Cell Biology, University of Montreal, Montreal, QC, Canada ´ *Corresponding author: Roger Lippe, [email protected] Enveloped viruses acquire their host-derived membrane at a variety of intracellular locations. Herpesviruses are complex entities that undergo several budding and fusion events during an infection. All members of this large family are believed to share a similar life cycle. However, they seemingly differ in terms of acquisition of their mature envelope. Herpes simplex virus is often believed to bud into an existing intracellular compartment, while the related cytomegalovirus may acquire its final envelope from a novel virus-induced assembly compartment. This review focuses on recent advances in the characterization of cellular compartment(s) potentially contributing to herpes virion final envelopment. It also examines the common points between seemingly distinct envelopment pathways and highlights the dynamic nature of intracellular compartments in the context of herpesvirus infections. Key words: assembly compartment, CMV, cytomegalovirus, egress, endosomes, ESCRT, herpesvirus, HSV, multivesicular bodies, PRV, TGN, VZV Received 24 May 2012, revised and accepted for publication 13 July 2012, uncorrected manuscript published online 17 July 2012, published online 6 August 2012

Host Compartment Identity Protein markers are commonly used to molecularly define intracellular compartments. As these markers play functional roles at these compartments, they also define molecular machineries operating at these sites. As shown in Figure 1, several other complementary tools are also available for the identification of intracellular compartments, based on morphology, subcellular localization, function, enzymatic activities, luminal pH, lipid composition, and in case of the endocytic route, the time it takes to transit from the cell surface (1,2). For instance, the trans Golgi network (TGN) is positive for the integral protein TGN46 in humans (TGN38 in rodents) and the lipid ceramide. It has a slightly acidic pH of 6.0–6.4 and harbors specific sugar modifying enzymes (e.g. sialyltransferase) (3–5). Furthermore, several cellular machineries are implicated in the transport of molecules to and

from the TGN, including the Rab subfamily of small GTPases (6). It can thus be identified using a variety of means.

Intracellular Compartments are Dynamic While molecular markers provide snap shots of intracellular compartments, they are in fact in a constant flux (1,2,7). For example, Golgi-resident enzymes are constantly recycled back to the Golgi/TGN when they escape (8). Many markers nonetheless primarily accumulate at unique sites, hence their diagnostic value (Figure 1). Some proteins are best used in combination with additional markers or considered in connection to time. For example, the transferrin receptor (TfR) accumulates at multiple sites, including the plasma membrane (PM), early endosomes (EE) and recycling endosomes (RE). However, it is a common marker of the endocytic pathway in pulse chase experiments that monitor its uptake from the cell surface (9). It is worth noting that internalized markers can label the EE, late endosomes (LE), lysosomes or sometimes even the TGN when allowed sufficient time or in sufficient concentration (10). This is particularly relevant as there is a direct link between EE and the TGN (8,11–15). Cellular membranes are also highly dynamic structures that are actively relocalized. It has been estimated that cells can internalize up to five times the equivalent of their PM every single hour (2). Thus the cell is far from static and many of its components are constantly reshuffled. Consequently, care needs to be exercised when defining specific cellular compartments. Viruses are strict intracellular pathogens that highjack various cellular pathways to complete their life cycles, infect other cells and spread within and between hosts. While individual viral proteins travel by the same pathways as host proteins, viral particle trafficking is a more complex matter. To understand how viruses use intracellular transport pathways and potentially discover novel antiviral drug targets, scientists face the challenge of identifying organelles that have been significantly altered by viruses (16,17).

Herpesviruses Interact with Multiple Cellular Compartments Herpesviruses are among the most complex animal viruses due to their intricate life cycles, large genome and complex composition (18). Eight different herpesviruses www.traffic.dk 1443

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Figure 1: Subcellular markers in uninfected cells. Organelles can be distinguished by the presence of several well-established markers (red), by their intraluminal pH (green), the kinetics to reach them from the cell surface (blue) and by interfering with proteins required for vesicle trafficking (black). However, these organelles interact, are highly dynamic, and markers can sometimes be detected on more than one compartment. Note that some endocytosed markers can eventually be recycled in the TGN if allowed sufficient time. EE, early endosomes; ER, endoplasmic reticulum; MVB, multivesicular bodies (also referred to as LE); RE, recycling endosomes; Ly, lysosomes; ESCRT, endosomal sorting complex required for transport; ST, sialyl-transferase.

infect humans and are subgrouped into three subfamilies, including Alphaherpesviruses [herpes simplex virus types 1 and 2 (HSV) as well as varicella-zoster virus (VZV)], Betaherpesviruses [human cytomegalovirus (HCMV) and roseoloviruses (HHV-6 and -7)] and Gammaherpesviruses (Epstein-Barr virus (EBV), Kaposi-sarcoma herpesvirus (KSHV)). They cause diseases ranging from cold sores to mononucleosis, B-cell lymphoma or encephalitis. Many additional members infect other species, for example, the well-studied pig pseudorabies virus (PRV), a non-human Alphaherpesvirus. Herpesviruses are thought to share their life cycle and their capacity to establish lifelong latent infections (18). Herpesvirus particles have diameters ranging from 120 to 300 nm and consist of several viral and cellular proteins that form the DNA-containing capsid, the cell-derived envelope (containing several transmembrane proteins required for attaching to and infecting cells) and the tegument (an intermediate protein layer-containing molecules required early in infection) (19,20). Herpesviruses enter cells by membrane fusion and the capsids reach the nucleus by recruiting molecular motors (16,21,22). They inject their genome into the nucleus where viral genome replication and transcription take place. New capsids are then assembled in the nucleus and incorporate the newly replicated viral genomes. These capsids, too big to travel through the nuclear pore (23,24), are believed to escape the nuclei by budding into the gap 1444

between the two nuclear membranes, a process referred to as primary envelopment (25,26). They are subsequently de-enveloped by fusion with the outer nuclear membrane. Once in the cytosol, these naked capsids travel to the site of final envelopment, where envelope proteins await and mature virions are assembled (27,28). Along this journey, tegument proteins are sequentially recruited to the capsid or interact with envelope proteins at the site of envelopment (25,26). The nature of the cellular compartment where final envelopment takes place is highly debated. Its identification is crucial since it is likely intimately linked to the mechanism of particle formation, which is so far poorly understood. Herpesviruses profoundly reorganize the cytoskeleton and intracellular compartments. The extent of rearrangements differs between cell types and some cell lines, such as 143B and MeWo, are somewhat more resistant, making them good tools for morphological studies (27). In this light, the identification of the final envelopment compartment is all the more challenging. This review focuses on recent advances in identifying cellular compartment(s) that contribute to the herpes virion final envelope. Given that many herpesviruses replicate very slowly or are predominantly latent, we will center our attention on HSV-1, PRV, VZV and HCMV.

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Envelopment of Herpesviruses

What Defines the Final Envelopment Site? Producing new virions at the site of final envelopment requires several events to occur. First, the DNA-containing capsid has to reach a membrane that necessarily contains all viral envelope proteins. Second, these proteins must be recruited and retained at the site of envelopment. Third, given that the tegument proteins form a layer between the capsid and envelope, any tegument proteins not previously bound to the viral capsids must also travel to the site of final envelopment. Fourth, membrane topology dictates that the newly wrapped virion must be surrounded by a second layer of membrane derived from the envelopment compartment or alternatively bud into the lumen of that compartment. Finally, blocking egress out of that compartment should trap the virus there if the virus enters its lumen. Any proposed envelopment site should fulfill these requirements.

Numerous Roads Lead to the TGN for Alphaherpesviruses The final envelopment of Alphaherpesviruses, a subclass of herpesviruses that includes HSV-1, PRV and VZV, seemingly differs from that of their Betaherpesvirus HCMV cousin. In the first case, the TGN appears to be an important site of envelopment (Figure 2). Capsids of several Alphaherpesviruses have indeed been identified

in structures morphologically resembling the TGN that are positive for the TGN markers TGN46, TGN38 or C6-NBDCer (29–32). These findings are corroborated by the lipid composition of extracellular virions reportedly resembling that of the TGN/Golgi (33). In addition, HSV-1 biochemically co-purifies with a TGN46-positive compartment and to a lesser extent with Rab5-positive endosomes (34). Using a synchronized infection and analysis of viral glycoproteins and established cellular markers, our lab highlighted the pivotal role played by the TGN, although that study could also not rule out a contribution by EE (27). Fulfilling yet another prediction, many of the Alphaherpesvirus glycoproteins and tegument proteins have been either detected at the TGN (27,28,35–38), found to precede the capsid at the TGN (27,38) or contain endocytic signals implied in TGN targeting (36,39–51), as reviewed elsewhere (52) Furthermore, blocking the biosynthetic pathway or redirecting virion components to the ER hampers their incorporation into mature virions and viral egress (53–55). Not surprisingly, inhibition of the cellular protein kinase D, a central player in the exit of cargo from the TGN, causes the accumulation of virions in TGNderived tubules and leads to reduced extracellular viral yields (56). Finally, myosin Va was reported to impact a post-TGN step (57). These findings fulfill the envelopment predictions stated above and point toward the TGN as a primary site of final envelopment for HSV, VZV and PRV. Although the bulk of the available data favors the TGN, two of the above studies could not formally

Figure 2: Model of Alphaherpesvirus final envelopment. Herpesvirus capsids, tegument and envelope proteins assemble on a compartment that shares markers of the TGN (shown in blue) in the case of Alphaherpesviruses (HSV, VZV and PRV). Following final envelopment, vesicles containing a virion leave the site of envelopment and are released from the cell by fusion with the PM. The host proteins that have been implicated in final envelopment and downstream egress steps are depicted in red. Note that this schematic does not take into consideration the significant reorganization of cellular architecture by herpesviruses, because it is not well defined. PKD, protein kinase D. See Figure 1 for other abbreviations.

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exclude EE as alternative sites for the envelopment of Alphaherpesviruses (27,34). Similarly, LE have also indirectly been implicated (58,59). However, not all the necessary viral glycoproteins, tegument proteins and capsids localize to these compartments, as would be expected if these two locations were significant contributors. Interestingly, several herpesviruses can induce autophagy and viral capsids can be engulfed by autophagosomes (60–62). Moreover, Rab1 has recently been linked to autophagosomes (63) and HSV-1 final envelopment (64). However, autophagosomes are unlikely to play an essential part in final envelopment, since autophagy inhibition does not influence viral replication (65). In conclusion, the TGN appears to be an important site of envelopment for Alphaherpesviruses. Despite a collection of mutant viruses with impaired envelopment (26), its mechanism is poorly understood. Over expression of dominant-negative mutants of the cellular ESCRT components Vps4 and Vps24 perturb HSV1 envelope protein gB trafficking and virus egress (58,59). Oddly, the ESCRT components ALIX and TSG101 are not involved in this process (66). The ESCRT machinery modulates multivesicular bodies (MVB) biogenesis in noninfected cells (67), but HSV-1 capsids have never been observed in that compartment. Similarly, it was recently shown that Rab1, a regulator of ER-to-Golgi transport, and Rab43, implicated in the integrity of the TGN, are involved in the final envelopment of HSV-1 (64). Given that ESCRT proteins, Rab1 and Rab43 act in different locations in noninfected cells, it is likely that the virus selectively recruits these components, much the same way HIV redirects ESCRT components to the PM for envelopment (68). It remains to be seen whether these cellular proteins act directly on envelopment or indirectly on the delivery of essential components to the site of final envelopment.

The Betaherpesvirus HCMV Redefines its Own Envelopment Site Several studies support a potential role of MVBs in HCMV envelopment. Although a role for Vps4 in HCMV final envelopment was initially ruled out using siRNA depletion (69), it was reported that dominant-negative mutants of Vps4 or CHMP1 do inhibit HCMV egress (70). Moreover, it was shown that a HCMV UL71 mutant alters MVB biogenesis and causes a delay in capsid envelopment (71). Finally, the HCMV envelope proteins UL33 and US27 were observed within MVB structures by electron microscopy and colocalized with LAMP1 and CD63 (72). However, HCMV has also been proposed to acquire its final envelope from the ER-Golgi intermediate compartment (73), postGolgi vacuoles (74) and LE (72) based on viral proteins present at those locations. Although HCMV induces autophagosomes much like its HSV-1 counterpart, neither virus has yet been observed in such structures (61). Though these potential envelopment sites were difficult to reconcile at first, it was suggested that the so-called 1446

HCMV assembly compartment is a virus-induced novel entity that is TGN46, Rab3 and ManII positive resulting from the reorganization of existing cellular compartments (74) (Figure 3). Similarly, Cepeda and colleagues proposed that the HCMV assembly compartment comprises both the TGN and endosomal markers TGN46, EEA1, CD63, TfR and M6PR, which are all present in mature virions (75). Thus, HCMV may reshuffle the cellular architecture to generate a unique re-envelopment site. Similar to Alphaherpesviruses, little is known about the mechanism of HCMV envelope acquisition. In addition to the ESCRT machinery, an eclectic set of host proteins has been implicated in HCMV egress. First, syntaxin 3 (Stx3), a SNARE involved in TGN-to-PM intracellular transport, accumulates in viral assembly compartments and hampers capsid envelopment (76). Second, the small GTPase Rab27a, a regulator of lysosome-related organelles secretion (77), is present in mature HCMV virions and modulates its envelopment (78). Third, HCMV gM interacts with the Rab11 effector FIP4, which is needed for optimal viral yields and the proper assembly of the envelopment compartment (79). Fourth, inhibition of PACS-1, a host protein recognizing acidic clusters in the endocytic pathway, impairs HCMV gB trafficking and viral production (80). Finally, Rab6 is needed for efficient release of infectious virions (81). This small GTPase is implicated in Golgi-to-ER and EE-to-TGN retrograde transport as well as Golgi-to-PM anterograde transport (82,83). Interestingly, Rab6 and Rab11 share the common effector Rab6 interactor protein 1 (R6IP1) that may be necessary for the fusion of MVBs and the RE compartments (84). As for HSV-1, it is unclear at present whether these host proteins impact the envelopment process per se and/or only indirectly affect the trafficking of viral particles by altering the delivery of essential components to the assembly site. It is also not yet clear if the association of the HCMV with all these markers is in a single compartment or represents different stages of envelope formation. A number of markers are indeed present at the assembly compartment but other components have not been documented there. Moreover, given the impact of HCMV on numerous intracellular compartments, the multiplicity of the cellular machineries implicated in HCMV final envelopment may reflect the need for multiple pathways to initiate or complete a cascade required to modify the compartments properly. Therefore, seemingly unrelated pathways may all impinge upon virus production with some directly involved and others only indirectly.

Reconciling HCMV with its Alphaherpesvirus Cousins? Differences between envelopment strategies employed by Alpha- and Betaherpesviruses like HCMV might reflect adaptations to their different host cell types. Moreover, the difficulty to identify the envelopment compartments

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Envelopment of Herpesviruses

Figure 3: Model of Betaherpesvirus final envelopment. Unlike Alphaherpesviruses, the compartment used for the final envelopment of HCMV is likely unique and is formed from extensively altered compartments in infected cells. In addition, most of the host proteins so far implicated in final envelopment and downstream egress steps (shown in red) are distinct from HSV-1 (see Figure 2). Once again, this schematic does not take into consideration the very significant reorganization of cellular architecture by the virus. STX3, Syntaxin 3; R6IP1, Rab6 interacting protein 1; FIP4, Rab11-interacting protein 4; PACS-1, phosphofurin acidic cluster sorting protein-1. See Figures 1 and 2 for additional abbreviations.

may partly result from the significant reorganization of intracellular compartments inflicted by herpesviruses. For instance, a comparative analysis of endocytic markers in uninfected and HCMV-infected cells showed that Rab5 and its effector EEA1 no longer colocalize with each other in infected cells, but rather with the LE markers LAMP1 and CD63 or the TGN marker p230 (85). This shows that these normally functionally linked markers are either no longer recruited to classical endosomes or that a radical reorganization of these intracellular compartments takes place to generate a unique compartment. This is consistent with HCMV envelopment likely taking place at a compartment that is specific to infected cells. Similarly, Alphaherpesviruses modulate intracellular compartments, albeit perhaps to a lesser extent than HCMV. During an HSV infection, both the Golgi and TGN fall apart and TGN markers can be found as far away as the nucleus, dispersed in the cytoplasm or at the PM (27,86–88). It was even suggested that HSV1 causes the dispersal of TGN membranes to form additional cytoplasmic compartments that may be used for envelopment (28). Thus both subfamilies do significantly re-organize intracellular compartments.

Conclusion and Future Directions The bulk of evidence currently suggests that the TGN contributes significantly to the envelopment of Alphaherpesviruses, whereas the Betaherpesvirus HCMV seems

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to build its own assembly compartment from existing organelles (Figures 2 and 3). Future studies will be needed to address the molecular machineries facilitating envelopment and determine to which extent the two subfamilies truly differ. Despite the implication of several cellular and viral proteins originating from distinct intracellular sites, it is presumably their recruitment and presence at high local concentrations at a given location that defines the site of assembly. It will thus be equally important to characterize the mechanism of recruitment and function of these molecules, which may constitute interesting antiviral drug targets. Furthermore, a better insight into how viruses highjack so many cellular components might expand our understanding of cellular trafficking events and organelle dynamics in times of cellular stress. One final unexplored possibility is that Herpesviruses might use different routes and sites for final envelopment depending on cell type, infection kinetics and/or virus load. For instance, it remains to be seen whether final envelopment of Alphaherpesviruses in neurons takes place at the TGN at its usual location near the nucleus, at a reorganized and/or redirected TGN down the axon, or at distinct site(s).

Acknowledgments This work was funded by the Canadian Institutes of Health Research (grant MOP 82921). K. R. is the recipient of a German Research Foundation postdoctoral fellowship (RA1608/4-1). The authors do not have any conflict of interest to declare.

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