Viral Tegument Protein Research Articles

Herpes Simplex Virus Utilizes the Large Secretory Vesicle Pathway for Anterograde Transport of Tegument and Envelope Proteins and for Viral Exocytosis from Growth Cones of Human Fetal Axons

Axonal transport of herpes simplex virus (HSV-1) is essential for viral infection and spread in the peripheral nervous system of the host. Therefore, the virus probably utilizes existing active transport and targeting mechanisms in neurons for virus assembly and spread from neurons to skin. In the present study, we used transmission immunoelectron microscopy to investigate the nature and origin of vesicles involved in the anterograde axonal transport of HSV-1 tegument and envelope proteins and of vesicles surrounding partially and fully enveloped capsids in growth cones. This study aimed to elucidate the mechanism of virus assembly and exit from axons of human fetal dorsal root ganglia neurons. We demonstrated that viral tegument and envelope proteins can travel in axons independently of viral capsids and were transported to the axon terminus in two types of transport vesicles, tubulovesicular membrane structures and large dense-cored vesicles. These vesicles and membrane carriers were derived from the trans-Golgi network (TGN) and contained key proteins, such as Rab3A, SNAP-25, GAP-43, and kinesin-1, involved in the secretory and exocytic pathways in axons. These proteins were also observed on fully and partially enveloped capsids in growth cones and on extracellular virions. Our findings provide further evidence to the subassembly model of separate transport in axons of unenveloped capsids from envelope and tegument proteins with final virus assembly occurring at the axon terminus. We postulate that HSV-1 capsids invaginate tegument- and envelope-bearing TGN-derived vesicles and utilize the large secretory vesicle pathway of exocytosis for exit from axons.

Herpes Simplex Virus Replication: Roles of Viral Proteins and Nucleoporins in Capsid-Nucleus Attachment

Replication of herpes simplex virus type 1 (HSV-1) involves a step in which a parental capsid docks onto a host nuclear pore complex (NPC). The viral genome then translocates through the nuclear pore into the nucleoplasm, where it is transcribed and replicated to propagate infection. We investigated the roles of viral and cellular proteins in the process of capsid-nucleus attachment. Vero cells were preloaded with antibodies specific for proteins of interest and infected with HSV-1 containing a green fluorescent protein-labeled capsid, and capsids bound to the nuclear surface were quantified by fluorescence microscopy. Results showed that nuclear capsid attachment was attenuated by antibodies specific for the viral tegument protein VP1/2 (UL36 gene) but not by similar antibodies specific for UL37 (a tegument protein), the major capsid protein (VP5), or VP23 (a minor capsid protein). Similar studies with antibodies specific for nucleoporins demonstrated attenuation by antibodies specific for Nup358 but not Nup214. The role of nucleoporins was further investigated with the use of small interfering RNA (siRNA). Capsid attachment to the nucleus was attenuated in cells treated with siRNA specific for either Nup214 or Nup358 but not TPR. The results are interpreted to suggest that VP1/2 is involved in specific attachment to the NPC and/or in migration of capsids to the nuclear surface. Capsids are suggested to attach to the NPC by way of the complex of Nup358 and Nup214, with high-resolution immunofluorescence studies favoring binding to Nup358.

Role of the UL25 Protein in Herpes Simplex Virus DNA Encapsidation

The herpes simplex virus protein UL25 attaches to the external vertices of herpes simplex virus type 1 capsids and is required for the stable packaging of viral DNA. To define regions of the protein important for viral replication and capsid attachment, the 580-amino-acid UL25 open reading frame was disrupted by transposon mutagenesis. The UL25 mutants were assayed for complementation of a UL25 deletion virus, and in vitro-synthesized protein was tested for binding to UL25-deficient capsids. Of the 11 mutants analyzed, 4 did not complement growth of the UL25 deletion mutant, and analysis of these and additional mutants in the capsid-binding assay demonstrated that UL25 amino acids 1 to 50 were sufficient for capsid binding. Several UL25 mutations were transferred into recombinant viruses to analyze the effect of the mutations on UL25 capsid binding and on DNA cleavage and packaging. Studies of these mutants demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids and that the C terminus is essential for DNA packaging and the production of infectious virus through its interactions with other viral packaging or tegument proteins. Analysis of viral DNA cleavage demonstrated that in the absence of a functional UL25 protein, aberrant cleavage takes place at the unique short end of the viral genome, resulting in truncated viral genomes that are not retained in capsids. Based on these observations, we propose a model where UL25 is required for the formation of DNA-containing capsids by acting to stabilize capsids that contain full-length viral genomes.

Characterization of White Spot Syndrome Virus Envelope Protein VP51A and Its Interaction with Viral Tegument Protein VP26

In this study, we characterize a novel white spot syndrome virus (WSSV) structural protein, VP51A (WSSV-TW open reading frame 294), identified from a previous mass spectrometry study. Temporal-transcription analysis showed that vp51A is expressed in the late stage of WSSV infection. Gene structure analysis showed that the transcription initiation site of vp51A was 135 bp upstream of the translation start codon. The poly(A) addition signal overlapped with the translation stop codon, TAA, and the poly(A) tail was 23 bp downstream of the TAA. Western blot analysis of viral protein fractions and immunoelectron microscopy both suggested that VP51A is a viral envelope protein. Western blotting of the total proteins extracted from WSSV virions detected a band that was close to the predicted 51-kDa mass, but the strongest signal was around 72 kDa. We concluded that this 72-kDa band was in fact the full-length VP51A protein. Membrane topology assays demonstrated that the VP51A 72-kDa protein is a type II transmembrane protein with a highly hydrophobic transmembrane domain on its N terminus and a C terminus that is exposed on the surface of the virion. Coimmunoprecipitation, colocalization, and yeast two-hybrid assays revealed that VP51A associated directly with VP26 and indirectly with VP28, with VP26 acting as a linker protein in the formation of a VP51A-VP26-VP28 complex.

Herpesvirus tegument protein activates NF-κB signaling through the TRAF6 adaptor protein

Viruses remodel the host cell to optimize their replication both by delivery of virion proteins into the cell and by de novo expression of viral proteins. The HSV particle contains several proteins that function to prepare the host cell for viral replication, including the VP16 transcriptional activator protein and virion host shutoff protein. HSV infection activates NF-kappaB pathways through Toll-like receptor (TLR) 2 and non-TLR pathways, and NF-kappaB activity is required for efficient viral replication. In a screen of the HSV proteome, we observed that the HSV U(L)37 tegument protein activates NF-kappaB signaling in a TLR2-independent manner. Expression of U(L)37 in transfected cells leads to IkappaB degradation and activation of both reporter genes and the endogenous IL-8 gene. This activation requires TNF receptor-associated factor 6 (TRAF6), and U(L)37 contains a TRAF6-binding domain that is required for interaction with TRAF6 and activation of NF-kappaB. A mutant virus encoding U(L)37 with an altered TRAF6-binding site shows reduced NF-kappaB activation in the early phase of infection. Therefore, the HSV U(L)37 virion structural protein can activate NF-kappaB through TRAF6. Activation of NF-kappaB by a virion tegument protein that is delivered into the host cell cytoplasm during viral entry represents a mechanism for activation of this pathway by a virus.

Multiple Functions for ORF75c in Murid Herpesvirus-4 Infection

All gamma-herpesviruses encode at least one homolog of the cellular enzyme formyl-glycineamide-phosphoribosyl-amidotransferase. Murid herpesvirus-4 (MuHV-4) encodes 3 (ORFs 75a, 75b and 75c), suggesting that at least some copies have acquired new functions. Here we show that the corresponding proteins are all present in virions and localize to infected cell nuclei. Despite these common features, ORFs 75a and 75b did not substitute functionally for a lack of ORF75c, as ORF75c virus knockouts were severely impaired for lytic replication in vitro and for host colonization in vivo. They showed 2 defects: incoming capsids failed to migrate to the nuclear margin following membrane fusion, and genomes that did reach the nucleus failed to initiate normal gene expression. The latter defect was associated with a failure of in-coming virions to disassemble PML bodies. The capsid transport deficit seemed to be functionally more important, since ORF75c− MuHV-4 infected both PML+ and PML− cells poorly. The original host enzyme has therefore evolved into a set of distinct and multi-functional viral tegument proteins. One important function is moving incoming capsids to the nuclear margin for viral genome delivery.

Tegument Proteins of Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a common, medically relevant human herpesvirus. The tegument layer of herpesvirus virions lies between the genome-containing capsids and the viral envelope. Proteins within the tegument layer of herpesviruses are released into the cell upon entry when the viral envelope fuses with the cell membrane. These proteins are fully formed and active and control viral entry, gene expression, and immune evasion. Most tegument proteins accumulate to high levels during later stages of infection, when they direct the assembly and egress of progeny virions. Thus, viral tegument proteins play critical roles at the very earliest and very last steps of the HCMV lytic replication cycle. This review summarizes HCMV tegument composition and structure as well as the known and speculated functions of viral tegument proteins. Important directions for future investigation and the challenges that lie ahead are identified and discussed.

Murine gammaherpesvirus 68 open reading frame 75c tegument protein induces the degradation of PML and is essential for production of infectious virus.

Promyelocytic Leukemia nuclear body (PML NB) proteins mediate an intrinsic cellular host defense response against virus infections. Herpesviruses express proteins that modulate PML or PML-associated proteins by a variety of strategies, including degradation of PML or relocalization of PML NB proteins. The consequences of PML-herpesvirus interactions during infection in vivo have yet to be investigated in detail, largely because of the species-specific tropism of many human herpesviruses. Murine gammaherpesvirus 68 (gammaHV68) is emerging as a suitable model to study basic biological questions of virus-host interactions because it naturally infects mice. Therefore, we sought to determine whether gammaHV68 targets PML NBs as part of its natural life cycle. We found that gammaHV68 induces PML degradation through a proteasome-dependent mechanism and that loss of PML results in more robust virus replication in mouse fibroblasts. Surprisingly, gammaHV68-mediated PML degradation was mediated by the virion tegument protein ORF75c, which shares homology with the cellular formylglycinamide ribotide amidotransferase enzyme. In addition, we show that ORF75c is essential for production of infectious virus. ORF75 homologs are conserved in all rhadinoviruses but so far have no assigned functions. Our studies shed light on a potential role for this unusual protein in rhadinovirus biology and suggest that gammaHV68 will be a useful model for investigation of PML-herpesvirus interactions in vivo.

Efficient secretion of the herpes simplex virus tegument protein VP22 from living mammalian cells

Many studies show that a tegument protein, VP22, of herpes simplex virus possesses an unusual capacity for intercellular trafficking, while several studies have reported that the intercellular trafficking was observed only in cells after fixation. Therefore, the trafficking ability in living cells remains controversial. To settle the question, we first examined secretion of VP22 in living cells. In this report, we fused VP22 with beta-galactosidase (betaGal) and investigated the secretion of VP22-betaGal in living cells by monitoring betaGal activity in the culture medium. Under our conditions, a significant amount of VP22-betaGal was detected in the culture medium, and it increased with time. Particularly, 6 days after transfection, 72% of all VP22-betaGal expressed was detected in the culture medium. Lactate dehydrogenase assays revealed that leakage of VP22-betaGal from damaged cells was not the main cause of the high level of secretion. We thus conclude that VP22 possesses a remarkable ability to be secreted from living cells.

Proteasome-dependent, ubiquitin-independent degradation of Daxx by the viral pp71 protein in human cytomegalovirus-infected cells

The cellular Daxx protein represses human cytomegalovirus (HCMV) gene expression from the major immediate early promoter. HCMV prevents Daxx-mediated silencing during lytic infection by delivering the viral pp71 tegument protein to the nucleus, where pp71 binds to and induces the proteasomal degradation of Daxx. In this study, we show that a functional ubiquitin pathway is not required for the proteasomal degradation of the endogenous Daxx protein by tegument-delivered pp71 in HCMV-infected cells, demonstrating that the pp71-mediated degradation of Daxx occurs through a proteasome-dependent, ubiquitin-independent pathway.

Expression of VCA (viral capsid antigen) and EBNA1 (Epstein–Barr‐virus‐encoded nuclear antigen 1) genes of Epstein–Barr virus in Pichia pastoris and application of the products in a screening test for patients with nasopharyngeal carcinoma

EBV (Epstein-Barr virus) serological tests have been used for many years as accessory diagnostic predictors of NPC (nasopharyngeal carcinoma). To date, IF (indirect immunofluorescence) assays still serve as the 'gold standard' for EBV serodiagnosis. However, IF assays are time-consuming, unsuitable for automatic handling and difficult to standardize. This makes their application in mass screening of populations inconvenient. Some of the technical difficulties associated with IF have been overcome by the development of specific ELISAs, but, at present, high sensitivity and specificity cannot be achieved simultaneously by using recombinant protein-based ELISAs, as the diagnostic value of different fragments of EBV in NPC is different. In an attempt to determine a suitable recombinant EBV protein for diagnostic purposes, fragments of EBV VCA (viral capsid antigen) and EBNA1 (Epstein-Barr-virus-encoded nuclear antigen 1) genes were expressed in the methylotrophic yeast Pichia pastoris, and a novel ELISA was established using P. pastoris-expressed VCA-BALF4 [aa (amino acids) 287-623; the BALF4 gene encodes the EBV glycoprotein gp125], EBNA1 (aa 390-641) and VCA-BFRF3 (the gene BFRF3 encodes a viral structural capsid protein or tegument protein VCA p18) proteins. Serum samples were collected from patients with NPC and healthy controls and were tested using this ELISA. The sensitivity of VCA-BFRF3, VCA-BALF4 and EBNA1 tests in the NPC sera were 65.0 (195/300), 76.3 (229/300) and 81.4% (244/300) respectively, whereas the specificity of normal individuals were 92 (460/500), 96 (480/500) and 95.8% (479/500). The optimum combination is VCA-BALF4 plus EBNA1, which identified 90.3% (271/300) of the NPC patients and had a specificity of 92.8% (464/500) for normal individuals. The results obtained from the evaluation of three antibodies to EBV as markers for detecting NPC suggests that a combination of EBNA1 (aa 390-641) and VCA-BALF4 (aa 287-623) assays would give better results in screening for NPC.

Betaherpesvirus-Conserved Cytomegalovirus Tegument Protein ppUL32 (pp150) Controls Cytoplasmic Events during Virion Maturation

The UL32 gene of human cytomegalovirus (CMV) encodes a prominent betaherpesvirus-conserved virion tegument protein, called pp150 (basic phosphoprotein/ppUL32), that accumulates within a cytoplasmic inclusion adjacent to the nucleus at late times during infection. Using a UL32 deletion mutant (DeltaUL32-BAC) (where BAC is bacterial artificial chromosome), we demonstrate that pp150 is critical for virion maturation in the cytoplasmic compartment. Cotransfection of a pp150 expression plasmid with DeltaUL32-BAC DNA led to complementation of the replication defect with focus formation due to secondary spread. Deletion of the amino terminus of pp150 or disruption of the betaherpesvirus conserved regions, CR1 and CR2, revealed these regions to be critical for replication. In contrast, deletion of the carboxyl terminus only partially compromised maturation while disruption of glycosylation sites had no effect. An African green monkey CMV UL32 homolog complemented DeltaUL32-BAC replication but murine CMV M32 failed to complement, consistent with evolutionary divergence of rodent and primate cytomegaloviruses. Infection with DeltaUL32-BAC showed normal expression of all kinetic classes of viral genes and replication of viral DNA, with accumulation of viral DNA-containing particles in the cytoplasm; however, mutant virus did not spread to adjacent cells. In contrast to this block in virion infectivity, cell-to-cell transfer of pp65-containing particles was observed, suggesting that release of dense bodies continued in the absence of pp150. These observations demonstrate that pp150 is critical for virion egress, possibly at the stage of final envelopment.

The Herpesvirus Capsid Surface Protein, VP26, and the Majority of the Tegument Proteins Are Dispensable for Capsid Transport toward the Nucleus

Upon entering a cell, alphaherpesvirus capsids are transported toward the minus ends of microtubules and ultimately deposit virus DNA within the host nucleus. The virus proteins that mediate this centripetal transport are unknown but are expected to be either viral tegument proteins, which are a group of capsid-associated proteins, or a surface component of the capsid itself. Starting with derivatives of pseudorabies virus that encode a fluorescent protein fused to a structural component of the virus, we have made a collection of 12 mutant viruses that lack either the VP26 capsid protein or an individual tegument protein. Using live-cell fluorescence microscopy, we tracked individual virus particles in axons following infection of primary sensory neurons. Quantitative analysis of the VP26-null virus indicates that this protein plays no observable role in capsid transport. Furthermore, viruses lacking tegument proteins that are nonessential for virus propagation in cell culture were also competent for axonal transport. These results indicate that a protein essential for viral propagation mediates transport of the capsid to the nucleus.

Epstein-Barr Virus Protein Kinase BGLF4 Is a Virion Tegument Protein That Dissociates from Virions in a Phosphorylation-Dependent Process and Phosphorylates the Viral Immediate-Early Protein BZLF1

Epstein-Barr virus (EBV) BGLF4 is a viral protein kinase that is expressed in the lytic phase of infection and is packaged in virions. We report here that BGLF4 is a tegument protein that dissociates from the virion in a phosphorylation-dependent process. We also present evidence that BGLF4 interacts with and phosphorylates BZLF1, a key viral regulator of lytic infection. These conclusions are based on the following observations. (i) In in vitro tegument release assays, a significant fraction of BGLF4 was released from virions in the presence of physiological NaCl concentrations. (ii) Addition of physiological concentrations of ATP and MgCl(2) to virions enhanced BGLF4 release, but phosphatase treatment of virions significantly reduced BGLF4 release. (iii) A recombinant protein containing a domain of BZLF1 was specifically phosphorylated by purified recombinant BGLF4 in vitro, and BGLF4 altered BZLF1 posttranslational modification in vivo. (iv) BZLF1 was specifically coimmunoprecipitated with BGLF4 in 12-O-tetradecanoylphorbol-13-acetate-treated B95-8 cells and in COS-1 cells transiently expressing both of these viral proteins. (v) BGLF4 and BZLF1 were colocalized in intranuclear globular structures, resembling the viral replication compartment, in Akata cells treated with anti-human immunoglobulin G. Our results suggest that BGLF4 functions not only in lytically infected cells by phosphorylating viral and cellular targets but also immediately after viral penetration like other herpesvirus tegument proteins.

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.

Phosphorylation of the Varicella-Zoster Virus (VZV) Major Transcriptional Regulatory Protein IE62 by the VZV Open Reading Frame 66 Protein Kinase

IE62, the major transcriptional regulatory protein encoded by varicella-zoster virus (VZV), is nuclear at early times of VZV infection but then becomes predominantly cytoplasmic as a result of expression of the protein kinase encoded by open reading frame 66 (ORF66). Cytoplasmic forms of IE62 are required for its inclusion as an abundant VZV virion tegument protein. Here we show that ORF66 directly phosphorylates IE62 at two residues, with phosphorylation at S686 being sufficient to regulate IE62 nuclear import. Phosphotryptic peptide analyses established an ORF66 kinase-mediated phosphorylation of the complete IE62 protein in transfected and VZV-infected cells. Using truncated and point-mutated IE62 peptides, ORF66-directed phosphorylation was mapped to residues S686 and S722, immediately downstream of the IE62 nuclear localization signal. An IE62 protein with an S686A mutation retained efficient nuclear import activity, even in the presence of functional ORF66 protein kinase, but an IE62 protein containing an S686D alteration was imported into the nucleus inefficiently. In contrast, the nuclear import of IE62 carrying an S722A mutation was still modulated by ORF66 expression, and IE62 with an S722D mutation was imported efficiently into the nucleus. An in vitro phosphorylation assay was developed using bacterially expressed IE62-maltose binding protein fusions as substrates for immunopurified ORF66 protein kinase from recombinant baculovirus-infected insect cells. ORF66 kinase phosphorylated the IE62 peptides, with similar specificities for residues S686 and S722. These results indicate that IE62 nuclear import is modulated as a result of direct phosphorylation of IE62 by ORF66 kinase. This represents an interaction that is, so far, unique among the alphaherpesviruses.

Transcripts of the human herpesvirus 8 genome in skin lesions and peripheral blood mononuclear cells of a patient with classic Kaposi's sarcoma.

We report a 75-year-old Japanese woman with classic Kaposi's sarcoma. PCR amplified human herpesvirus 8 (HHV-8) DNA sequences from her skin lesions and peripheral blood mononuclear cells (PBMC), but not her plasma, saliva or urine. An antibody test against HHV-8 lytic antigens was positive. Immunohistochemical staining detected latent antigen. There was no evidence of HHV-8 infection in her husband, sister or daughter. Genes coding for HHV-8-encoded viral interleukin-6, viral macrophage inflammatory protein I, viral G protein-coupled receptor, viral cyclin D and viral Bcl-2 were expressed to the same degree in both her skin lesion and PBMC. Latency-associated T0.7 mRNA and HHV-8-encoded viral tegument protein genes were expressed in her PBMC at levels lower than in the skin lesions. Based on the gene expression profile, we concluded that lytic HHV-8 infection was present in her skin lesions and PBMC.

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.

Senile plaques in Alzheimer’s diseased brains: Possible association of β-amyloid with herpes simplex virus type 1 (HSV-1) L-particles

The characteristic insoluble, senile (neuritic) plaques found extracellularly in brains of patients with Alzheimer's disease (AD) contain the fibrillar form of beta-amyloid (Abeta42). A substantial proportion of autopsied elderly brains have demonstrated DNA evidence of herpes simplex virus type 1 (HSV-1) infiltration. HSV-1-infected cells produce significant quantities of non-infectious, non-DNA-containing light particles (L-particles) comprised of viral envelope and tegument proteins. HSV-induced L-particles can be exocytosed out of their host cells. This report advances the hypothesis that (1) Abeta binds to L-particles; (2) Abeta permeabilizes L-particles, destroying the integrity of the envelope and allowing the contained tegument proteins to spill into the extracellular space; and (3) these events are followed by a conformational shift of Abeta into its fibrillar form, physically trapping the L-particle-derived substances and resulting in the plaques characteristic of AD. These hypotheses are supported by reports of biomolecular changes and pathophysiologies which have been simultaneously observed in both AD- and HSV-infected brains.

Herpes Simplex Virus Tegument Protein VP22 Contains an Internal VP16 Interaction Domain and a C-Terminal Domain That Are Both Required for VP22 Assembly into the Virus Particle

Many steps along the herpesvirus assembly and maturation pathway remain unclear. In particular, the acquisition of the virus tegument is a poorly understood process, and the molecular interactions involved in tegument assembly have not yet been defined. Previously we have shown that the two major herpes simplex virus tegument proteins VP22 and VP16 are able to interact, although the relevance of this to virus assembly is not clear. Here we have constructed a number of recombinant viruses expressing N- and C-terminal truncations of VP22 and have used them to identify regions of the protein involved in its assembly into the virus structure. Analysis of the packaging of these VP22 variants into extracellular virions revealed that the C terminus of VP22 is absolutely required for this process, with removal of the C-terminal 89 residues abrogating its incorporation. However, while these 89 residues alone were sufficient for specific incorporation of small amounts of VP22 into the tegument, efficient packaging of VP22 to the levels of full-length protein required an additional 52 residues of the protein. Coimmunoprecipitation assays indicated that these 52 residues also contained the interaction domain for VP16. Furthermore, analysis of the subcellular localization of the mutant forms of VP22 revealed that only those truncations that were efficiently assembled formed characteristic cytoplasmic trafficking complexes, suggesting that these complexes may represent the cellular location for VP22 assembly into the virus. Taken together, these results suggest that there are two determinants involved in the packaging of VP22-a C-terminal domain and an internal VP16 interaction domain, both of which are required for the efficient recruitment of VP22 to sites of virus assembly.