The UL13 and US3 Protein Kinases of Herpes Simplex Virus 1 Cooperate to Promote the Assembly and Release of Mature,Infectious Virions

Herpes simplex virus type 1 (HSV-1) encodes two bona fide serine/threonine protein kinases, the US3 and UL13 gene products. HSV-1 ΔUS3 mutants replicate with wild-type efficiency in cultured cells, and HSV-1 ΔUL13 mutants exhibit <10-fold reduction in infectious viral titers. Given these modest phenotypes, it remains unclear how the US3 and UL13 protein kinases contribute to HSV-1 replication. In the current study, we designed a panel of HSV-1 mutants, in which portions of UL13 and US3 genes were replaced by expression cassettes encoding mCherry protein or green fluorescent protein (GFP), respectively, and analyzed DNA replication, protein expression, and spread of these mutants in several cell types. Loss of US3 function alone had largely negligible effect on viral DNA accumulation, gene expression, virion release, and spread. Loss of UL13 function alone also had no appreciable effects on viral DNA levels. However, loss of UL13 function did result in a measurable decrease in the steady-state levels of two viral glycoproteins (gC and gD), release of total and infectious virions, and viral spread. Disruption of both genes did not affect the accumulation of viral DNA, but resulted in further reduction in gC and gD steady-state levels, and attenuation of viral spread and infectious virion release. These data show that the UL13 kinase plays an important role in the late phase of HSV-1 infection, likely by affecting virion assembly and/or release. Moreover, the data suggest that the combined activities of the US3 and UL13 protein kinases are critical to the efficient assembly and release of infectious virions from HSV-1-infected cells.     

Varicella-Zoster Virus ORF12 Protein Activates the Phosphatidylinositol 3-Kinase/Akt Pathway To Regulate Cell Cycle Progression

Varicella-zoster virus (VZV) activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and alters cell cycle progression, but the viral protein(s) responsible for these activities is unknown. We previously reported that the VZV open reading frame 12 (ORF12) protein triggers phosphorylation of ERK. Here, we demonstrate that the VZV ORF12 protein also activates the PI3K/Akt pathway to regulate cell cycle progression. Transfection of cells with a plasmid expressing the ORF12 protein induced phosphorylation of Akt, which was dependent on PI3K. Infection of cells with wild-type VZV triggered phosphorylation of Akt, while infection with an ORF12 deletion mutant induced less phosphorylated Akt. The activation of Akt by ORF12 protein was associated with its binding to the p85 subunit of PI3K. Infection of cells with wild-type VZV resulted in increased levels of cyclin B1, cyclin D3, and phosphorylated glycogen synthase kinase 3β (GSK-3β), while infection with the ORF12 deletion mutant induced lower levels of these proteins. Wild-type VZV infection reduced the G₁ phase cell population and increased the M phase cell population, while infection with the ORF12 deletion mutant had a reduced effect on the G₁ and M phase populations. Inhibition of Akt activity with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 reduced the G₁ and M phase differences observed in cells infected with wild-type and ORF12 mutant viruses. In conclusion, we have found that the VZV ORF12 protein activates the PI3K/Akt pathway to regulate cell cycle progression. Since VZV replicates in both dividing (e.g., keratinocytes) and nondividing (neurons) cells, the ability of the VZV ORF12 protein to regulate the cell cycle is likely important for VZV replication in various cell types in the body.     

The Herpes Simplex Virus Type 1 UL17 Gene Encodes Virion Tegument Proteins That Are Required for Cleavage and Packaging of Viral DNA

Previous studies have suggested that the U_L17 gene of herpes simplex virus type 1 (HSV-1) is essential for virus replication. In this study, viral mutants incorporating either a lacZ expression cassette in place of 1,490 bp of the 2,109-bp U_L17 open reading frame [HSV-1(ΔU_L17)] or a DNA oligomer containing an in-frame stop codon inserted 778 bp from the 5′ end of the U_L17 open reading frame [HSV-1(U_L17-stop)] were plaque purified on engineered cell lines containing the U_L17 gene. A virus derived from HSV-1(U_L17-stop) but containing a restored U_L17 gene was also constructed and was designated HSV-1(U_L17-restored). The latter virus formed plaques and cleaved genomic viral DNA in a manner indistinguishable from wild-type virus. Neither HSV-1(ΔU_L17) nor HSV-1(U_L17-stop) formed plaques or produced infectious progeny when propagated on noncomplementing Vero cells. Furthermore, genomic end-specific restriction fragments were not detected in DNA purified from noncomplementing cells infected with HSV-1(ΔU_L17) or HSV-1(U_L17-stop), whereas end-specific fragments were readily detected when the viruses were propagated on complementing cells. Electron micrographs of thin sections of cells infected with HSV-1(ΔU_L17) or HSV-1(U_L17-stop) illustrated that empty capsids accumulated in the nuclei of Vero cells, whereas DNA-containing capsids accumulated in the nuclei of complementing cells and enveloped virions were found in the cytoplasm and extracellular space. Additionally, protein profiles of capsids purified from cells infected with HSV-1(ΔU_L17) compared to wild-type virus show no detectable differences. These data indicate that the U_L17 gene is essential for virus replication and is required for cleavage and packaging of viral DNA. To characterize the U_L17 gene product, an anti-U_L17 rabbit polyclonal antiserum was produced. The antiserum reacted strongly with a major protein of apparent M_r 77,000 and weakly with a protein of apparent M_r 72,000 in wild-type infected cell lysates and in virions. Bands of similar sizes were also detected in electrophoretically separated tegument fractions of virions and light particles and yielded tryptic peptides of masses characteristic of the predicted U_L17 protein. We therefore conclude that the U_L17 gene products are associated with the virion tegument and note that they are the first tegument-associated proteins shown to be required for cleavage and packaging of viral DNA.     

The Herpes Simplex Virus Type 1 Cleavage/Packaging Protein,UL32, Is Involved in Efficient Localization of Capsids to Replication Compartments

Six genes, including UL32, have been implicated in the cleavage and packaging of herpesvirus DNA into preassembled capsids. We have isolated a UL32 insertion mutant which is capable of near-wild-type levels of viral DNA synthesis; however, the mutant virus is unable to cleave and package viral DNA, consistent with the phenotype of a previously isolated temperature-sensitive herpes simplex virus type 1 mutant, tsN20 (P. A. Schaffer, G. M. Aron, N. Biswal, and M. Benyesh-Melnick, Virology 52:57–71, 1973). A polyclonal antibody which recognizes UL32 was previously used by Chang et al. (Y. E. Chang, A. P. Poon, and B. Roizman, J. Virol. 70:3938–3946, 1996) to demonstrate that UL32 accumulates predominantly in the cytoplasm of infected cells. In this report, a functional epitope-tagged version of UL32 showed that while UL32 is predominantly cytoplasmic, some nuclear staining which colocalizes with the major DNA binding protein (ICP8, UL29) in replication compartments can be detected. We have also used a monoclonal antibody (5C) specific for the hexon form of major capsid protein VP5 to study the distribution of capsids during infection. In cells infected with wild-type KOS (6 and 8 h postinfection), 5C staining patterns indicate that capsids are present in nuclei within replication compartments. These results suggest that cleavage and packaging occur in replication compartments at least at 6 and 8 h postinfection. Cells infected with the UL32 mutant exhibit a hexon staining pattern which is more diffusely distributed throughout the nucleus and which is not restricted to replication compartments. We propose that UL32 may play a role in “bringing” preassembled capsids to the sites of DNA packaging and that the failure to localize to replication compartments may explain the cleavage/packaging defect exhibited by this mutant. These results suggest that the UL32 protein is required at a step distinct from those at which other cleavage and packaging proteins are required and may be involved in the correct localization of capsids within infected cells.During infection of cells with herpes simplex virus type 1 (HSV-1), the large concatemeric products of DNA replication are cleaved to unit length and packaged into preassembled capsids. Capsids are icosahedral structures composed of 150 hexons and 12 pentons. Three types of capsids (A, B, and C) can be isolated from infected cells by velocity centrifugation (20). C capsids contain the viral DNA genome; B capsids contain the scaffolding protein; and A capsids contain neither DNA nor the scaffolding protein. Pulse chase experiments with another alphaherpesvirus, equine herpesvirus 1, indicate that at least some B capsids can package DNA and mature into infectious virions, while A capsids cannot (46). By analogy with the bacteriophages, these results suggest that B capsids represent procapsids which are intermediates in the packaging process. However, a new intermediate in the assembly process has recently been identified (41, 62). These newly identified capsid forms observed in in vitro assembly extracts have the same protein content as B capsids but are more spherical; these capsids are unstable and adopt the more angular form characteristic of B capsids after prolonged incubation in vitro. These results suggest that the unstable spherical forms may represent the true procapsid intermediate (41, 62).In many bacteriophages, the procapsid contains at least three essential components: an icosahedrally arranged protein shell, an internal scaffold, and a dodecameric ring called the portal vertex through or around which the phage DNA is taken up (8, 11, 18). For HSV-1, the outer shell is composed of four proteins: the major capsid protein, VP5; a small protein bound to hexons, VP26; and a triplex structure made up of heterotrimers of VP19C and VP23 (reviewed in reference 56). VP24, VP21, and VP22a are found in the interior of the capsid and are encoded by overlapping genes UL26 and UL26.5; VP21 and VP22a are present in B but not A or C capsids and are considered to make up the internal scaffold (reviewed in reference 56). Although bacteriophages contain a portal vertex, no such structure has been observed in HSV-1 capsids. Whether the herpesviruses have a unique portal vertex through which viral DNA is taken up is unclear; it is possible that this type of unique vertex is only needed in viruses which have a tail. Capsids indistinguishable from those isolated from HSV-1-infected cells have been observed in extracts from insect cells infected with recombinant baculoviruses bearing HSV-1 capsid genes (42, 60). Therefore, it is clear that these proteins are sufficient for capsid assembly in vitro; however, it is not known whether capsids formed in vitro are competent for DNA uptake. It is possible that minor components of capsids play important roles in genome encapsidation.In addition to the capsid proteins, at least six genes are essential for the encapsidation of viral DNA: the UL6, UL15, UL25, UL28, UL32, and UL33 genes. Temperature-sensitive (ts) strains with mutations in these genes have similar phenotypes, in that viral DNA can be replicated but not cleaved and packaged (1, 2, 4, 6, 48, 51, 54, 55, 66). Strains with null mutations in the UL6, UL15, UL25, UL28, and UL33 genes have been isolated and characterized, thereby confirming the roles of these genes in cleavage and packaging (5, 27, 37, 45, 59, 68). Despite the identification of these required genes, the mechanism by which viral DNA is cleaved and packaged is not understood, nor has the role of any of the gene products been determined. The UL6 and UL25 proteins have been detected in A, B, and C capsids as well as in virions (3, 28, 37, 44); however, the precise role of these two proteins in capsids remains to be determined.A ts UL32 mutant, tsN20, defective in cleavage and packaging, has been reported previously (51). Because mutants with lesions resulting in temperature sensitivity are often prone to problems associated with incomplete penetrance at the nonpermissive temperature, we isolated a UL32 insertion mutant, hr64. Characterization of hr64 confirms that UL32 is essential for cleavage and packaging. Previous studies demonstrated that UL32 localizes to the cytoplasm of infected cells (13). We have used a functional epitope-tagged version of UL32 to confirm that in infected cells, this protein is mainly cytoplasmic, although some nuclear staining was observed.HSV-1 DNA replication occurs in globular nuclear domains termed “replication compartments” initially identified by ICP8 (UL29) staining patterns in an immunofluorescence assay (49). All seven replication proteins have now been localized within replication compartments (10, 24, 29–31, 43) as has regulatory protein ICP4 (26, 50). Ward et al. have recently reported that at late times after infection (18 h), capsids accumulate in the nucleus in regions distinct from replication compartments (64). These authors suggest that these regions represent assembly stations in which DNA is packaged. We report herein, however, that at 6 and 8 h postinfection, capsids colocalize with ICP8 in replication compartments. This suggests that at these early times, cleavage and packaging occur within replication compartments. Furthermore, we report that in cells infected with the UL32 mutant virus, capsids are distributed throughout the nucleus, accumulating in regions outside the replication compartments. This suggests that UL32 may play a role in the efficient localization of capsids in infected cells.     

Phosphorylation of the UL31 Protein of Herpes Simplex Virus 1 by the US3-Encoded Kinase Regulates Localization of the Nuclear Envelopment Complex and Egress of Nucleocapsids

Herpes simplex virus 1 nucleocapsids bud through the inner nuclear membrane (INM) into the perinuclear space to obtain a primary viral envelope. This process requires a protein complex at the INM composed of the U_L31 and U_L34 gene products. While it is clear that the viral kinase encoded by the U_S3 gene regulates the localization of pU_L31/pU_L34 within the INM, the molecular mechanism by which this is accomplished remains enigmatic. Here, we have determined the following. (i) The N terminus of pU_L31 is indispensable for the protein''s normal function and contains up to six serines that are phosphorylated by the U_S3 kinase during infection. (ii) Phosphorylation at these six serines was not essential for a productive infection but was required for optimal viral growth kinetics. (iii) In the presence of active U_S3 kinase, changing the serines to alanine caused the pU_L31/pU_L34 complex to aggregate at the nuclear rim and caused some virions to accumulate aberrantly in herniations of the nuclear membrane, much as in cells infected with a U_S3 kinase-dead mutant. (iv) The replacement of the six serines of pU_L31 with glutamic acid largely restored the smooth distribution of pU_L34/pU_L31 at the nuclear membrane and precluded the accumulation of virions in herniations whether or not U_S3 kinase was active but also precluded the optimal primary envelopment of nucleocapsids. These observations indicate that the phosphorylation of pU_L31 by pU_S3 represents an important regulatory event in the virion egress pathway that can account for much of pU_S3''s role in nuclear egress. The data also suggest that the dynamics of pU_L31 phosphorylation modulate both the primary envelopment and the subsequent fusion of the nascent virion envelope with the outer nuclear membrane.The U_L31 and U_L34 proteins of herpes simplex virus 1 (HSV-1) form a complex that accumulates at the inner nuclear membrane (INM) of infected cells (26, 27). This complex is essential for the budding of nucleocapsids through the INM into the perinuclear space (26, 28). pU_L34 is a type 2 integral membrane protein with a 247-amino-acid nucleoplasmic domain that binds pU_L31 and holds the latter in close approximation to the INM (16, 19, 26, 31, 36, 37). Both proteins become incorporated into nascent virions, indicating that they directly or indirectly interact with nucleocapsids during the budding event (27). Interestingly, the coexpression of the pseudorabies virus homologs of HSV pU_L31 and pU_L34 are sufficient to induce budding from the INM in the absence of other viral proteins (13).The most prominent model of nuclear egress proposes that the step following primary envelopment involves the fusion of the perinuclear virion envelope with the outer nuclear membrane (ONM), allowing subsequent steps in which the deenveloped capsid engages budding sites in the Golgi or trans-Golgi network (20, 32). The U_S3 protein is a promiscuous kinase that phosphorylates pU_L31, pU_L34, and several other viral and cellular components (1, 2, 5, 11, 15, 21-23, 25). In the absence of pU_S3 kinase activity, (i) virions accumulate within distensions of the perinuclear space that herniate into the nucleoplasm (14, 27, 29), (ii) the pU_L31/pU_L34 complex is mislocalized at the nuclear rim from a smooth pattern to discrete foci that accumulate adjacent to nuclear membrane herniations (12, 14, 27, 29), and (iii) the onset of infectious virus production is delayed (21, 29).Aberrant accumulations of perinuclear virions similar to those observed in cells infected with U_S3 kinase-dead viruses have been observed in cells infected with viruses lacking the capacity to produce glycoproteins H and B (gH and gB, respectively) (8). Because these proteins are required for fusion with the plasma membrane or endocytic vesicles during HSV entry (3, 4, 9, 10, 18, 30, 33), it has been proposed that the accumulation of perinuclear virions in the absence of gH and gB reflects a failure in the apparatus that normally mediates the fusion between the nascent virion envelope and the ONM (8). By extension of this hypothesis, pU_S3 might act to trigger or otherwise regulate this perinuclear fusion event.The substrate(s) of the pU_S3 kinase responsible for the altered localization of the pU_L31/pU_L34 complex and the aberrant accumulation of perinuclear virions were heretofore unknown. In one study to identify such a substrate, it was determined that precluding the phosphorylation of pU_L34 was not responsible for the nuclear egress defects induced by the absence of pU_S3 or its kinase activity (29). The current study was therefore undertaken to investigate the hypothesis that the pU_S3-mediated phosphorylation of pU_L31 is critical to regulate nuclear egress. The presented evidence indicates that aspects of the U_S3 kinase-dead phenotype, including the retention of virions in the perinuclear space, the mislocalization of the pU_L31/pU_L34 complex, and the delayed onset of virus replication, can be replicated by precluding pU_L31 phosphorylation in the presence or absence of pU_S3 kinase activity. The data also suggest that the dynamic phosphorylation of pU_L31 is important during the primary envelopment of nucleocapsids.     

Attenuation of the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway by Porphyromonas gingivalis Gingipains RgpA,RgpB, and Kgp

Porphyromonas gingivalis is a major pathogen of periodontal diseases, including periodontitis. We have investigated the effect of P. gingivalis infection on the PI3K/Akt (protein kinase B) signaling pathway in gingival epithelial cells. Here, we found that live P. gingivalis, but not heat-killed P. gingivalis, reduced Akt phosphorylation at both Thr-308 and Ser-473, which implies a decrease in Akt activity. Actually, PI3K, which is upstream of Akt, was also inactivated by P. gingivalis. Furthermore, glycogen synthase kinase 3α/β, mammalian target of rapamycin, and Bad, which are downstream proteins in the PI3K/Akt cascade, were also dephosphorylated, a phenomenon consistent with Akt inactivation by P. gingivalis. However, these events did not require direct interaction between bacteria and host cells and were independent of P. gingivalis invasion into the cells. The use of gingipain-specific inhibitors and a gingipain-deficient P. gingivalis mutant KDP136 revealed that the gingipains and their protease activities were essential for the inactivation of PI3K and Akt. The associations between the PI3K regulatory subunit p85α and membrane proteins were disrupted by wild-type P. gingivalis. Moreover, PDK1 translocation to the plasma membrane was reduced by wild-type P. gingivalis, but not KDP136, indicating little production of phosphatidylinositol 3,4,5-triphosphate by PI3K. Therefore, it is likely that PI3K failed to transmit homeostatic extracellular stimuli to intracellular signaling pathways by gingipains. Taken together, our findings indicate that P. gingivalis attenuates the PI3K/Akt signaling pathway via the proteolytic effects of gingipains, resulting in the dysregulation of PI3K/Akt-dependent cellular functions and the destruction of epithelial barriers.     

Herpes Simplex Virus 1 Glycoprotein M and the Membrane-Associated Protein UL11 Are Required for Virus-Induced Cell Fusion and Efficient Virus Entry

Herpes simplex virus 1 (HSV-1) facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl-terminal 28 amino acids of gB or the dominant syncytial mutation in gK (Ala to Val at amino acid 40) was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletion substantially affected gB, gC, gD, gE, and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells more slowly than their parental wild-type virus strain. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread.     

US3 Protein Kinase of Herpes Simplex Virus 1 Blocks Caspase 3 Activation Induced by the Products of US1.5 and UL13 Genes and Modulates Expression of Transduced US1.5 Open Reading Frame in a Cell Type-Specific Manner

The coding domain of the herpes simplex virus type 1 (HSV-1) alpha22 gene encodes two proteins, the 420-amino-acid infected-cell protein 22 (ICP22) and U(S)1.5, a protein colinear with the carboxyl-terminal domain of ICP22. In HSV-1-infected cells, ICP22 and U(S)1.5 are extensively modified by the U(L)13 and U(S)3 viral protein kinases. In this report, we show that in contrast to other viral proteins defined by their properties as alpha proteins, U(S)1.5 becomes detectable and accumulated only at late times after infection. Moreover, significantly more U(S)1.5 protein accumulated in cells infected with a mutant lacking the U(L)13 gene than in cells infected with wild-type virus. To define the role of viral protein kinases on the accumulation of U(S)1.5 protein, rabbit skin cells or Vero cells were exposed to recombinant baculoviruses that expressed U(S)1.5, U(L)13, or U(S)3 proteins under a human cytomegalovirus immediate-early promoter. The results were as follows. (i) Accumulation of the U(S)1.5 protein was reduced by concurrent expression of the U(L)13 protein kinase and augmented by concurrent expression of the U(S)3 protein kinase. The magnitude of the reduction or increase in the accumulation of the U(S)1.5 protein was cell type dependent. The effect of U(L)13 kinase appears to be specific inasmuch as it did not affect the accumulation of glycoprotein D in cells doubly infected by recombinant baculoviruses expressing these genes. (ii) The reduction in accumulation of the U(S)1.5 protein was partially due to proteasome-dependent degradation. (iii) Both U(S)1.5 and U(L)13 proteins activated caspase 3, indicative of programmed cell death. (iv) Concurrent expression of the U(S)3 protein kinase blocked activation of caspase 3. The results are concordant with those published elsewhere (J. Munger and B. Roizman, Proc. Natl. Acad. Sci. USA 98:10410-10415, 2001) that the U(S)3 protein kinase can block apoptosis by degradation or posttranslational modification of BAD.     

Herpes Simplex Virus DNA Cleavage and Packaging: Association of Multiple Forms of UL15-Encoded Proteins with B Capsids Requires at Least the UL6, UL17, and UL28 Genes

The U_L15 gene of herpes simplex virus (HSV) is one of several genes required for the packaging of viral DNA into intranuclear B capsids to produce C capsids that become enveloped at the inner nuclear membrane. A rabbit antiserum directed against U_L15-encoded protein recognized three proteins with apparent M_rs of 79,000, 80,000, and 83,000 in highly purified B capsids. The 83,000-M_r protein was detected in type C capsids and comigrated with the product of a U_L15 cDNA transcribed and translated in vitro. The 83,000- and 80,000-M_r proteins were readily detected in purified virions. Inasmuch as (i) none of these proteins were detectable in capsids purified from cells infected with HSV-1(ΔU_L15), a virus lacking an intact U_L15 gene, and (ii) corresponding proteins in capsids purified from cells infected with a recombinant virus [HSV-1(R7244), containing a 20-codon tag at the 3′ end of U_L15] were decreased in electrophoretic mobility relative to the wild-type proteins, we conclude that the proteins with apparent M_rs of 83,000, 80,000, and 79,000 are products of U_L15 with identical C termini. The 79,000-, 80,000-, and 83,000-M_r proteins remained associated with B capsids in the presence of 0.5 M guanidine HCl and remained detectable in capsids treated with 2.0 M guanidine HCl and lacking proteins associated with the capsid core. These data, therefore, indicate that U_L15-encoded proteins are integral components of B capsids. Only the 83,000-M_r protein was detected in B capsids purified from cells infected with viruses lacking the U_L6, U_L17, or U_L28 genes, which are required for DNA cleavage and packaging, suggesting that capsid association of the 80,000- and 79,000-M_r proteins requires intact cleavage and packaging machinery. These data, therefore, indicate that capsid association of the 80,000- and 79,000-M_r U_L15-encoded proteins reflects a previously unrecognized step in the DNA cleavage and packaging reaction.     

Tsg101 Interacts with Herpes Simplex Virus 1 VP1/2 and Is a Substrate of VP1/2 Ubiquitin-Specific Protease Domain Activity

Ubiquitination/deubiquitination of key factors represent crucial steps in the biogenesis of multivesicular body (MVB) and sorting of transmembrane proteins. We and others previously demonstrated that MVB is involved in herpes simplex virus 1 (HSV-1) envelopment and budding. Here, we report that the HSV-1 large tegument protein, VP1/2, interacts with and regulates the ubiquitination of Tsg101, a cellular protein essential in MVB formation, thus identifying the first cellular substrate of a herpesviral deubiquitinating enzyme.