Nuclear localizations of the herpes simplex virus type 1 tegument proteins VP13/14, vhs, and VP16 precede VP22-dependent microtubule reorganization and VP22 nuclear import

Herpes simplex virus type 1 (HSV-1) induces microtubule reorganization beginning at approximately 9 h postinfection (hpi), and this correlates with the nuclear localization of the tegument protein VP22. Thus, the active retention of this major virion component by cytoskeletal structures may function to regulate its subcellular localization (A. Kotsakis, L. E. Pomeranz, A. Blouin, and J. A. Blaho, J. Virol. 75:8697-8711, 2001). The goal of this study was to determine whether the subcellular localization patterns of other HSV-1 tegument proteins are similar to that observed with VP22. To address this, we performed a series of indirect immunofluorescence analyses using synchronously infected cells. We observed that tegument proteins VP13/14, vhs, and VP16 localized to the nucleus as early as 5 hpi and were concentrated in nuclei by 9 hpi, which differed from that seen with VP22. Microtubule reorganization was delayed during infection with HSV-1(RF177), a recombinant virus that does not produce full-length VP22. These infected cells did not begin to lose microtubule-organizing centers until 13 hpi. Repair of the unique long 49 (UL49) locus in HSV-1(RF177) yielded HSV-1(RF177R). Microtubule reorganization in HSV-1(RF177R)-infected cells occurred with the same kinetics as HSV-1(F). Acetylated tubulin remained unchanged during infection with either HSV-1(F) or HSV-1(RF177). Thus, while alpha-tubulin reorganized during infection, acetylated tubulin was stable, and the absence of full-length VP22 did not affect this stability. Our findings indicate that the nuclear localizations of tegument proteins VP13/14, VP16, and vhs do not appear to require HSV-1-induced microtubule reorganization. We conclude that full-length VP22 is needed for optimal microtubule reorganization during infection. This implies that VP22 mainly functions to reorganize microtubules later, rather than earlier, in infection. That acetylated tubulin does not undergo restructuring during VP22-dependent, virus-induced microtubule reorganization suggests that it plays a role in stabilizing the infected cells. Our results emphasize that VP22 likely plays a key role in cellular cytopathology during HSV-1 infection.     

Characterization of a UL49-null mutant: VP22 of herpes simplex virus type 1 facilitates viral spread in cultured cells and the mouse cornea

Herpes simplex virus type 1 (HSV-1) virions, like those of all herpesviruses, contain a proteinaceous layer termed the tegument that lies between the nucleocapsid and viral envelope. The HSV-1 tegument is composed of at least 20 different viral proteins of various stoichiometries. VP22, the product of the U(L)49 gene, is one of the most abundant tegument proteins and is conserved among the alphaherpesviruses. Although a number of interesting biological properties have been attributed to VP22, its role in HSV-1 infection is not well understood. In the present study we have generated both a U(L)49-null virus and its genetic repair and characterized their growth in both cultured cells and the mouse cornea. While single-step growth analyses indicated that VP22 is dispensable for virus replication at high multiplicities of infection (MOIs), analyses of plaque morphology and intra- and extracellular multistep growth identified a role for VP22 in viral spread during HSV-1 infection at low MOIs. Specifically, VP22 was not required for either virion infectivity or cell-cell spread but was required for accumulation of extracellular virus to wild-type levels. We found that the absence of VP22 also affected virion composition. Intracellular virions generated by the U(L)49-null virus contained reduced amounts of ICP0 and glycoproteins E and D compared to those generated by the wild-type and U(L)49-repaired viruses. In addition, viral spread in the mouse cornea was significantly reduced upon infection with the U(L)49-null virus compared to infection with the wild-type and U(L)49-repaired viruses, identifying a role for VP22 in viral spread in vivo as well as in vitro.     

Analysis of the interaction between the essential herpes simplex virus 1 tegument proteins VP16 and VP1/2

The incorporation of tegument proteins into the herpes simplex virus 1 (HSV-1) virion during virion assembly is thought to be a complex, multistage process occurring via numerous interactions between the tegument and the capsid, within the tegument, and between the tegument and the envelope. Here, we set out to examine if the direct interaction between two essential tegument proteins VP1/2 and VP16 is required for connecting the inner tegument with the outer tegument. By using glutathione S-transferase (GST) pulldowns, we identified an essential role of lysine 343 in VP16, mutation of which to a neutral amino acid abrogated the interaction between VP1/2 and VP16. When the K343A substitution was inserted into the gene encoding VP16 (UL48) of the viral genome, HSV-1 replicated successfully although its growth was delayed, and final titers were reduced compared to titers of wild-type virus. Surprisingly, the mutated VP16 was incorporated into virions at levels similar to those of wild-type VP16. However, the analysis of VP16 on cytoplasmic capsids by fluorescence microscopy showed that VP16 associated with cytoplasmic capsids less efficiently when the VP16-VP1/2 interaction was inhibited. This implies that the direct interaction between VP1/2 and VP16 is important for the efficiency/timing of viral assembly but is not essential for HSV-1 replication in cell culture. These data also support the notion that the incorporation of tegument proteins into the herpesviruses is a very complex process with significant redundancy.     

Temporal regulation of herpes simplex virus type 2 VP22 expression and phosphorylation

The VP22 protein of herpes simplex virus type 2 (HSV-2) is a major component of the virion tegument. Previous work with HSV-1 indicated that VP22 is phosphorylated during infection, and phosphorylation may play a role in modulating VP22 localization in infected cells. It is not clear, however, when phosphorylation occurs in infected cells or how it is regulated. Less is known about the synthesis and phosphorylation of HSV-2 VP22. To study the complete biosynthetic history of HSV-2 VP22, we generated a monoclonal antibody to the carboxy terminus of VP22. Using immunoprecipitation and Western blot analyses, we show that HSV-2 VP22 can be found in three distinct isoforms in infected cells, two of which are phosphorylated. Like HSV-1 VP22, HSV-2 VP22 is synthesized ca. 4 h after infection, and the isoform later incorporated into virions is hypophosphorylated. In addition, we demonstrate for the first time (i) that newly synthesized VP22 is phosphorylated rapidly after synthesis, (ii) that this phosphorylation occurs in a virus-dependent manner, (iii) that the HSV-2 kinase UL13 is capable of inducing phosphorylation of VP22 in the absence of other viral proteins, (iv) that phosphorylated VP22 is very stable in infected cells, (v) that phosphorylated isoforms of VP22 are gradually dephosphorylated late in infection to produce the virion tegument form, and (vi) that this dephosphorylation occurs independently of viral DNA replication or virion assembly. These results indicate that HSV-2 VP22 is a stable protein that undergoes highly regulated, virus-dependent phosphorylation events in infected cells.     

Modified VP22 Localizes to the Cell Nucleus during Synchronized Herpes Simplex Virus Type 1 Infection

The UL49 gene product (VP22) of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) is a virion phosphoprotein which accumulates inside infected cells at late stages of infection. We previously (J. A. Blaho, C. Mitchell, and B. Roizman, J. Biol. Chem. 269:17401-17410, 1994) discovered that the form of VP22 packaged into infectious virions differed from VP22 extracted from infected-cell nuclei in that the virion-associated form had a higher electrophoretic mobility in denaturing gels. Based on these results, we proposed that VP22 in virions was "undermodified" in some way. The goal of this study is to document the biological and biochemical properties of VP22 throughout the entire course of a productive HSV-1 infection. We now report the following. (i) VP22 found in infected cells is distributed in at least three distinct subcellular localizations, which we define as cytoplasmic, diffuse, and nuclear, as measured by indirect immunofluorescence. (ii) Using a synchronized infection system, we determined that VP22 exists predominantly in the cytoplasm early in infection and accumulates in the nucleus late in infection. (iii) While cytoplasmic VP22 colocalizes with the HSV-1 glycoprotein D early in infection, the nuclear form of VP22 is not restricted to replication compartments which accumulate ICP4. (iv) VP22 migrates as at least three unique electrophoretic species in denaturing sodium dodecyl sulfate-DATD-polyacrylamide gels. VP22a, VP22b, and VP22c have high, intermediate, and low mobility, respectively. (v) The relative distribution of the various forms of VP22 derived from infected whole-cell extracts varies during the course of infection such that low-mobility species predominate at early times and high-mobility forms accumulate later. (vi) The highest-mobility forms of VP22 partition with the cytoplasmic fraction of infected cells, while the lowest-mobility forms are associated with the nuclear fraction. (vii) Finally, full-length VP22 which partitions in the nucleus incorporates radiolabel from [32P]orthophosphate whereas cytoplasmic VP22 does not. Based on these results, we conclude that modification of VP22 coincides with its appearance in the nucleus during the course of productive HSV-1 infection.     

Deletion of the herpes simplex virus VP22-encoding gene (UL49) alters the expression, localization, and virion incorporation of ICP0

The role of the herpes simplex virus tegument protein VP22 is not yet known. Here we describe the characterization of a virus in which the entire VP22 open reading frame has been deleted. We show that VP22 is not essential for virus growth but that virus lacking VP22 (Delta22) displays a cell-specific replication defect in epithelial MDBK cells. Virus particles assembled in the absence of VP22 show few obvious differences to wild-type (WT) particles, except for a moderate reduction in glycoproteins gD and gB. In addition, the Delta22 virus exhibits a general delay in the initiation of virus protein synthesis, but this is not due to a glycoprotein-related defect in virus entry. Intriguingly, however, the absence of VP22 has an obvious effect on the intracellular level of the immediate-early (IE) protein ICP0. Moreover, following translocation from the nucleus to the cytoplasm, ICP0 is unable to localize to the characteristic cytoplasmic sites observed in a WT infection. We demonstrate that, in WT-infected cells, VP22 and ICP0 are concentrated in the same cytoplasmic sites. Furthermore, we show that, while ICP0 and ICP4 are components of WT extracellular virions, the altered localization of ICP0 in the cytoplasm of Delta22-infected cells correlates with an absence of both ICP0 and ICP4 from Delta22 virions. Hence, while a role has not yet been defined for virion IE proteins in virus infection, our results suggest that their incorporation is a specific event requiring the tegument protein VP22. This report provides the first direct evidence that VP22 influences virus assembly.     

The Pattern of Tegument-Capsid Interaction in the Herpes Simplex Virus Type 1 Virion Is Not Influenced by the Small Hexon-Associated Protein VP26

Examination of the three-dimensional structure of intact herpes simplex virus type 1 (HSV-1) virions had revealed that the icosahedrally symmetrical interaction between the tegument and capsid involves the pentons but not the hexons (Z. H. Zhou, D. H. Chen, J. Jakana, F. J. Rixon, and W. Chiu, J. Virol. 73:3210-3218, 1999). To account for this, we postulated that the presence of the small capsid protein, VP26, on top of the hexons was masking potential binding sites and preventing tegument attachment. We have now tested this hypothesis by determining the structure of virions lacking VP26. Apart from the obvious absence of VP26 from the capsids, the structures of the VP26 minus and wild-type virions were essentially identical. Notably, they showed the same tegument attachment patterns, thereby demonstrating that VP26 is not responsible for the divergent tegument binding properties of pentons and hexons.     

Phosphorylation of Structural Components Promotes Dissociation of the Herpes Simplex Virus Type 1 Tegument

The role of phosphorylation in the dissociation of structural components of the herpes simplex virus type 1 (HSV-1) tegument was investigated, using an in vitro assay. Addition of physiological concentrations of ATP and magnesium to wild-type virions in the presence of detergent promoted the release of VP13/14 and VP22. VP1/2 and the UL13 protein kinase were not significantly solubilized. However, using a virus with an inactivated UL13 protein, we found that the release of VP22 was severely impaired. Addition of casein kinase II (CKII) to UL13 mutant virions promoted VP22 release. Heat inactivation of virions or addition of phosphatase inhibited the release of both proteins. Incorporation of radiolabeled ATP into the assay demonstrated the phosphorylation of VP1/2, VP13/14, VP16, and VP22. Incubation of detergent-purified, heat-inactivated capsid-tegument with recombinant kinases showed VP1/2 phosphorylation by CKII, VP13/14 phosphorylation by CKII, protein kinase A (PKA), and PKC, VP16 phosphorylation by PKA, and VP22 phosphorylation by CKII and PKC. Proteolytic mapping and phosphoamino acid analysis of phosphorylated VP22 correlated with previously published work. The phosphorylation of virion-associated VP13/14, VP16, and VP22 was demonstrated in cells infected in the presence of cycloheximide. Use of equine herpesvirus 1 in the in vitro release assay resulted in the enhanced release of VP10, the homolog of HSV-1 VP13/14. These results suggest that the dissociation of major tegument proteins from alphaherpesvirus virions in infected cells may be initiated by phosphorylation events mediated by both virion-associated and cellular kinases.     

Simultaneous tracking of capsid, tegument, and envelope protein localization in living cells infected with triply fluorescent herpes simplex virus 1

We report here the construction of a triply fluorescent-tagged herpes simplex virus 1 (HSV-1) expressing capsid protein VP26, tegument protein VP22, and envelope protein gB as fusion proteins with monomeric yellow, red, and cyan fluorescent proteins, respectively. The recombinant virus enabled us to monitor the dynamics of these capsid, tegument, and envelope proteins simultaneously in the same live HSV-1-infected cells and to visualize single extracellular virions with three different fluorescent emissions. In Vero cells infected by the triply fluorescent virus, multiple cytoplasmic compartments were found to be induced close to the basal surfaces of the infected cells (the adhesion surfaces of the infected cells on the solid growth substrate). Major capsid, tegument, and envelope proteins accumulated and colocalized in the compartments, as did marker proteins for the trans-Golgi network (TGN) which has been implicated to be the site of HSV-1 secondary envelopment. Moreover, formation of the compartments was correlated with the dynamic redistribution of the TGN proteins induced by HSV-1 infection. These results suggest that HSV-1 infection causes redistribution of TGN membranes to form multiple cytoplasmic compartments, possibly for optimal secondary envelopment. This is the first real evidence for the assembly of all three types of herpesvirus proteins-capsid, tegument, and envelope membrane proteins-in TGN.     

Varicella-zoster virus (VZV) open reading frame 10 protein, the homolog of the essential herpes simplex virus protein VP16, is dispensable for VZV replication in vitro.

Varicella-zoster virus (VZV) open reading frame 10 (ORF10) protein in the homolog of the herpes simplex virus type 1 (HSV-1) protein VP16. VZV ORF10 transactivates the VZV IE62 gene and is a tegument protein present in the virion. HSV-1 VP16, a potent transactivator of HSV-1 immediate-early genes and tegument protein, is essential for HSV-1 replication in vitro. To determine whether VZV ORF10 is required for viral replication in vitro, we constructed two VZV mutants which were unable to express ORF10. One mutant had a stop codon after the 61st codon of the ORF10 gene, and the other mutant was deleted for all but the last five codons of the gene. Both VZV mutants grew in cell culture to titers similar to that of the parental virus. To determine whether HSV-1 VP16 alters the growth of VZV, we constructed a VZV mutant in which VP16 was inserted in place of ORF10. Using immune electron microscopy, we found that HSV-1 VP16 was present in the tegument of the recombinant VZV virions. The VZV VP16 substitution mutant produced smaller plaques and grew to a lower titer than parental virus. Thus, VZV ORF10 is not required for growth of the virus in vitro, and substitution of HSV-1 VP16 for VZV ORF10 impairs the growth of VZV.     

Herpes simplex virus tegument protein VP16 is a component of primary enveloped virions

Immunogold electron microscopy was used to determine whether the tegument proteins VP13/14, VP22, and VP16 of herpes simplex virus type 1 (HSV1) are components of primary enveloped virions. Whereas VP13/14 and VP22 were not detected in virus particles in the perinuclear space and were present in only mature extracellular virions, VP16 was acquired prior to primary envelopment of the virus at the inner nuclear membrane. This finding highlights potential similarities and differences between HSV1 and the related alphaherpesvirus, pseudorabies virus, in which the homologues of all three of these tegument proteins are not incorporated into the virion until secondary envelopment.     

In rat dorsal root ganglion neurons,herpes simplex virus type 1 tegument forms in the cytoplasm of the cell body

The herpes simplex virus type 1 (HSV-1) tegument is the least understood component of the virion, and the mechanism of tegument assembly and incorporation into virions during viral egress has not yet been elucidated. In the present study, the addition of tegument proteins (VP13/14, VP16, VP22, and US9) and envelope glycoproteins (gD and gH) to herpes simplex virions in the cell body of rat dorsal root ganglion neurons was examined by immunoelectron microscopy. All tegument proteins were detected diffusely spread in the nucleus within 10 to 12 h and, at these times, nucleocapsids were observed budding from the nucleus. The majority (96%) of these nucleocapsids had no detectable label for tegument and glycoproteins despite the presence of tegument proteins in the nucleus and glycoproteins adjacent to the nuclear membrane. Immunolabeling for tegument proteins and glycoproteins was found abundantly in the cytoplasm of the cell body in multiple discrete vesicular areas: on unenveloped, enveloped, or partially enveloped capsids adjacent to these vesicles and in extracellular virions. These vesicles and intracytoplasmic and extracellular virions also labeled with Golgi markers, giantin, mannosidase II, and TGN38. Treatment with brefeldin A from 2 to 24 h postinfection markedly inhibited incorporation into virions of VP22 and US9 but to a lesser degree with VP16 and VP13/14. These results suggest that, in the cell body of neurons, most tegument proteins are incorporated into unenveloped nucleocapsids prior to envelopment in the Golgi and the trans-Golgi network. These findings give further support to the deenvelopment-reenvelopment hypothesis for viral egress. Finally, the addition of tegument proteins to unenveloped nucleocapsids in the cell body allows access to these unenveloped nucleocapsids to one of two pathways: egress through the cell body or transport into the axon.     

Actin is a component of the compensation mechanism in pseudorabies virus virions lacking the major tegument protein VP22

Despite being a major component of the pseudorabies virus tegument, VP22 is not required for PRV replication, virulence, or neuroinvasion (T. del Rio, H. C. Werner, and L. W. Enquist, J. Virol. 76:774-782, 2002). In the absence of VP22, tegument assembly compensates in a limited fashion with increased incorporation of cellular actin. Infection of epithelial cell lines expressing fluorescent actin fusion proteins resulted in the incorporation of filamentous and nonfilamentous actin into individual virions that were predominately light, noninfectious particles. We conclude that cellular actin is incorporated in the tegument of wild-type virions and is part of a compensation mechanism for VP22-null virions.     

A Network of Protein Interactions around the Herpes Simplex Virus Tegument Protein VP22

Assembly of the herpesvirus tegument is poorly understood but is believed to involve interactions between outer tegument proteins and the cytoplasmic domains of envelope glycoproteins. Here, we present the detailed characterization of a multicomponent glycoprotein-tegument complex found in herpes simplex virus 1 (HSV-1)-infected cells. We demonstrate that the tegument protein VP22 bridges a complex between glycoprotein E (gE) and glycoprotein M (gM). Glycoprotein I (gI), the known binding partner of gE, is also recruited into this gE-VP22-gM complex but is not required for its formation. Exclusion of the glycoproteins gB and gD and VP22''s major binding partner VP16 demonstrates that recruitment of virion components into this complex is highly selective. The immediate-early protein ICP0, which requires VP22 for packaging into the virion, is also assembled into this gE-VP22-gM-gI complex in a VP22-dependent fashion. Although subcomplexes containing VP22 and ICP0 can be formed when either gE or gM are absent, optimal complex formation requires both glycoproteins. Furthermore, and in line with complex formation, neither of these glycoproteins is individually required for VP22 or ICP0 packaging into the virion, but deletion of gE and gM greatly reduces assembly of both VP22 and ICP0. Double deletion of gE and gM also results in small plaque size, reduced virus yield, and defective secondary envelopment, similar to the phenotype previously shown for pseudorabies virus. Hence, we suggest that optimal gE-VP22-gM-gI-ICP0 complex formation correlates with efficient virus morphogenesis and spread. These data give novel insights into the poorly understood process of tegument acquisition.     

Ultrastructural visualization of individual tegument protein dissociation during entry of herpes simplex virus 1 into human and rat dorsal root ganglion neurons

Herpes simplex virus 1 (HSV-1) enters neurons primarily by fusion of the viral envelope with the host cell plasma membrane, leading to the release of the capsid into the cytosol. The capsid travels via microtubule-mediated retrograde transport to the nuclear membrane, where the viral DNA is released for replication in the nucleus. In the present study, the composition and kinetics of incoming HSV-1 capsids during entry and retrograde transport in axons of human fetal and dissociated rat dorsal root ganglia (DRG) neurons were examined by wide-field deconvolution microscopy and transmission immunoelectron microscopy (TIEM). We show that HSV-1 tegument proteins, including VP16, VP22, most pUL37, and some pUL36, dissociated from the incoming virions. The inner tegument proteins, including pUL36 and some pUL37, remained associated with the capsid during virus entry and transit to the nucleus in the neuronal cell body. By TIEM, a progressive loss of tegument proteins, including VP16, VP22, most pUL37, and some pUL36, was observed, with most of the tegument dissociating at the plasma membrane of the axons and the neuronal cell body. Further dissociation occurred within the axons and the cytosol as the capsids moved to the nucleus, resulting in the release of free tegument proteins, especially VP16, VP22, pUL37, and some pUL36, into the cytosol. This study elucidates ultrastructurally the composition of HSV-1 capsids that encounter the microtubules in the core of human axons and the complement of free tegument proteins released into the cytosol during virus entry.     

Live-cell analysis of a green fluorescent protein-tagged herpes simplex virus infection

Many stages of the herpes simplex virus maturation pathway have not yet been defined. In particular, little is known about the assembly of the virion tegument compartment and its subsequent incorporation into maturing virus particles. Here we describe the construction of a herpes simplex virus type 1 (HSV-1) recombinant in which we have replaced the gene encoding a major tegument protein, VP22, with a gene expressing a green fluorescent protein (GFP)-VP22 fusion protein (GFP-22). We show that this virus has growth properties identical to those of the parental virus and that newly synthesized GFP-22 is detectable in live cells as early as 3 h postinfection. Moreover, we show that GFP-22 is incorporated into the HSV-1 virion as efficiently as VP22, resulting in particles which are visible by fluorescence microscopy. Consequently, we have used time lapse confocal microscopy to monitor GFP-22 in live-cell infection, and we present time lapse animations of GFP-22 localization throughout the virus life cycle. These animations demonstrate that GFP-22 is present in a diffuse cytoplasmic location when it is initially expressed but evolves into particulate material which travels through an exclusively cytoplasmic pathway to the cell periphery. In this way, we have for the first time visualized the trafficking of a herpesvirus structural component within live, infected cells.     

Phosphorylation of the herpes simplex virus tegument protein VP22 has no effect on incorporation of VP22 into the virus but is involved in optimal expression and virion packaging of ICP0

Herpes simplex virus VP22 is a major tegument protein of unknown function. Very recently, we reported that the predominant effect of deleting the VP22 gene was on the expression, localization, and virion incorporation of ICP0. In addition, the Delta22 virus replicated poorly in epithelial MDBK cells. We have also previously shown that VP22 interacts with the tegument protein VP16 and the cellular microtubule network. While the majority of VP22 in infected cells is highly phosphorylated, the nonphosphorylated form of VP22 is the predominant species in the virion, suggesting a differential requirement for phosphorylation through virus replication. Hence, to study the significance of VP22 phosphorylation, we have now constructed two recombinant viruses expressing green fluorescent protein-VP22 (G22) in which the previously identified serine phosphorylation sites have been mutated either to alanine to abolish the phosphorylation status of VP22 (G22P-) or to glutamic acid to mimic permanent phosphorylation (G22P+). Localization studies indicated that the G22P- protein associated tightly with microtubules in some infected cells, suggesting that VP22 phosphorylation may control its interaction with the microtubule network. By contrast, VP22 phosphorylation had no effect on its ability to interact with VP16 and, importantly, had no effect on the relative packaging of VP22. Intriguingly, virion packaging of ICP0 was reduced in the G22P+ virus while ICP0 expression was reduced in the G22P- virus, suggesting that these two ICP0 defects, previously observed in the Delta22 virus, were attributable to different forms of VP22. Furthermore, the Delta22 virus replication defect in MDBK cells correlated with the expression of constitutively charged VP22 in the G22P+ virus. Taken together, these results suggest an important role for VP22 phosphorylation in its relationship with ICP0.