Packaging of genomic and amplicon DNA by the herpes simplex virus type 1 UL25-null mutant KUL25NS

The herpes simplex virus type 1 (HSV-1) mutant KUL25NS, containing a null mutation within the UL25 gene, was isolated and characterized by McNab and coworkers (A. R. McNab, P. Desai, S. Person, L. L. Roof, D. R. Thomsen, W. W. Newcomb, J. C. Brown, and F. L. Homa, J. Virol. 72:1060-1070, 1998). This mutant was able to cleave the concatemeric products of viral DNA replication into monomeric units, but in contrast to wild-type (wt) HSV-1, they were degraded by DNase treatment, indicating that they were not stably packaged into virus capsids. I have examined the packaging of the KUL25NS genome and an HSV-1 amplicon in cells infected with the mutant virus. In contrast to the previous results, a low level of KUL25NS DNA was resistant to DNase digestion, indicating that it was retained in capsids. The proportion of this packaged DNA present as full-length genomes was much lower than in cells infected by wt HSV-1, and there was a significant overrepresentation of the long terminus and underrepresentation of the short terminus. KUL25NS was less impaired in stably packaging amplicon DNA than in packaging its own genome, and the packaged molecules contained approximately equimolar amounts of the two terminal fragments. Below about 100 kbp, the packaged amplicon molecules exhibited an abundance and size distribution similar to those generated using wt HSV-1 as a helper, but the mutant was relatively impaired in packaging longer amplicon molecules. Both packaged genomic and amplicon DNAs were retained in the nuclei of KUL25NS-infected cells. These results suggest that the UL25 protein may play an important role during the later stages of the head-filling process, prior to release of capsids into the cytoplasm.     

Characterization of VP22 in herpes simplex virus-infected cells

We examine biochemical characteristics of the herpes simplex virus (HSV) tegument protein VP22 by gel filtration, glycerol sedimentation, and chemical cross-linking experiments and use time course radiolabeling and immunoprecipitation assays to analyze its synthesis and interaction with other infected-cell proteins. VP22 was expressed as a delayed early protein with optimal synthesis requiring DNA replication. In immunoprecipitation assays, VP22 was found in association with several additional proteins including VP16 and a kinase activity likely to be that of UL13. Furthermore, in sizing chromatography experiments, VP22 was present in several higher-order complexes in infected cells. From gel filtration analysis the major form of VP22 migrated with a molecular mass of approximately 160 kDa, consistent with its presence as a tetramer, or a dimer complexed with other proteins, with a fraction of the protein migrating at larger molecular mass. In vitro-synthesized VP22 sedimented in a size range consistent with a mixture of tetramers and dimers. Short N- or C-terminal deletions resulted in migration almost exclusively as dimers, indicating that VP22, in the absence of additional virus-encoded proteins, could form higher-order assemblies, most likely tetramers, but that both N-and C-terminal determinants were required for stabilizing such assemblies. Consistent with this we found that isolated proteins encompassing either the N-terminal or C-terminal region of VP22 sedimented as dimers, and that the purified C-terminal domain could be cross-linked into dimeric structures. These results are discussed with regard to possible virus and host interactions involved in VP22 recruitment into virus particles.     

Varicella-zoster virus gene 51 complements a herpes simplex virus type 1 UL9 null mutant.

Varicella-zoster virus (VZV) gene 51 encodes a protein which is homologous to UL9, the origin of DNA replication-binding protein of herpes simplex virus type 1. No genetic information is available on VZV gene 51, but its product has been shown to bind to virtually the same recognition sequence as does UL9 (D. Chen and P. D. Olivo, J. Virol. 68:3841-3849, 1994; N. D. Stow, H. M. Weir, and E. C. Stow, Virology 177:570-577, 1990). We report here that gene 51 can complement a UL9 null mutant (hr94) (A. K. Malik, R. Martinez, L. Muncy, E. P. Carmichael, and S. K. Weller, Virology 190:702-715, 1992), but at a level which is only 20% of that of UL9. Quantitation of viral DNA synthesis suggests that this phenotype is due to a defect in viral DNA synthesis. Regardless, the ability of VZV gene 51 to complement UL9 suggests that alphaherpesviruses have a highly conserved mechanism of initiation of viral DNA synthesis.     

Membrane association of VP22, a herpes simplex virus type 1 tegument protein

Tegument proteins of herpes simplex virus type 1 (HSV-1) are hypothesized to contain the functional information required for the budding or envelopment process proposed to occur at cytoplasmic compartments of the host cell. One of the most abundant tegument proteins of HSV-1 is the U(L)49 gene product, VP22, a 38-kDa protein of unknown function. To study its subcellular localization, a VP22-green fluorescent protein chimera was expressed in transfected human melanoma (A7) cells. In the absence of other HSV-1 proteins, VP22 localizes to acidic compartments of the cell that may include the trans-Golgi network (TGN), suggesting that this protein is membrane associated. Membrane pelleting and membrane flotation assays confirmed that VP22 partitions with the cellular membrane fraction. Through truncation mutagenesis, we determined that the membrane association of VP22 is a property attributed to amino acids 120 to 225 of this 301-amino-acid protein. The above results demonstrate that VP22 contains specific information required for targeting to membranes of acidic compartments of the cell which may be derived from the TGN, suggesting a potential role for VP22 during tegumentation and/or final envelopment.     

Microtubule reorganization during herpes simplex virus type 1 infection facilitates the nuclear localization of VP22, a major virion tegument protein

Full-length VP22 is necessary for efficient spread of herpes simplex virus type 1 (HSV-1) from cell to cell during the course of productive infection. VP22 is a virion phosphoprotein, and its nuclear localization initiates between 5 and 7 h postinfection (hpi) during the course of synchronized infection. The goal of this study was to determine which features of HSV-1 infection function to regulate the translocation of VP22 into the nucleus. We report the following. (i) HSV-1(F)-induced microtubule rearrangement occurred in infected Vero cells by 13 hpi and was characterized by the loss of obvious microtubule organizing centers (MtOCs). Reformed MtOCs were detected at 25 hpi. (ii) VP22 was observed in the cytoplasm of cells prior to microtubule rearrangement and localized in the nucleus following the process. (iii) Stabilization of microtubules by the addition of taxol increased the accumulation of VP22 in the cytoplasm either during infection or in cells expressing VP22 in the absence of other viral proteins. (iv) While VP22 localized to the nuclei of cells treated with the microtubule depolymerizing agent nocodazole, either taxol or nocodazole treatment prevented optimal HSV-1(F) replication in Vero cells. (v) VP22 migration to the nucleus occurred in the presence of phosphonoacetic acid, indicating that viral DNA and true late protein synthesis were not required for its translocation. Based on these results, we conclude that (iv) microtubule reorganization during HSV-1 infection facilitates the nuclear localization of VP22.     

Glutamine deprivation causes enhanced plating efficiency of a herpes simplex virus type 1 ICP0-null mutant

Isoleucine deprivation of cellular monolayers prior to infection has been reported to result in partial complementation of a herpes simplex virus type 1 (HSV-1) ICP0 null (ICP0⁻) mutant. We now report that glutamine deprivation alone is able to enhance the plating efficiency of an ICP0⁻ virus and that isoleucine deprivation has little or no effect. Because a low glutamine level is associated with stress and because stress is known to induce reactivation, low levels of glutamine may be relevant to the reactivation of HSV-1 from latency. Additionally, we demonstrate that arginine and methionine deprivation result in partial complementation of the ICP0⁻ virus.     

Characterization of a temperature-sensitive mutant of the UL15 open reading frame of herpes simplex virus 1.

The UL15 gene of herpes simplex virus 1 consists of two exons and is highly conserved among the herpesviruses sequenced to date. Other than its homology to a phage protein involved in the packaging of DNA, nothing is known of its function. This report concerns the isolation of a temperature-sensitive mutant with a mutation mapping in the UL15 open reading frame. Cells infected with the parent, mutant, and rescued viruses all make DNA at the nonpermissive temperature. Direct analyses of the DNA and electron microscopic studies indicate that although viral DNA is made, it is not packaged into capsids present in nuclei. These studies suggest that UL15 may be involved in the packaging of viral DNA.     

Intercellular trafficking of herpes simplex virus type 2 UL14 deletion mutant proteins

The UL14 gene product of herpes simplex virus is a 32kDa protein expressed late in infection and is a minor component of the virion tegument. We recently showed that the wild-type UL14 protein has heat shock protein (HSP)-like and/or molecular chaperone-like functions. In this study, the intracellular localization of UL14 wild-type and deletion mutant proteins was examined in transfected cells by immunofluorescence. We found that N-terminus deleted but not wild-type/C-terminus deleted mutant proteins showed a significant number of cytoplasmic, multi-cellular stains in transfected Vero cells. The effect was greatly intensified by subjecting cells to heat shock at 43 degrees C, whereas it was obstructed by treatment with the microfilament-disrupting drug cytochalasin D. The staining patterns of UL14 antigen-positive cells after heat shock suggested a cell-to-cell spread of the protein. Although the mechanism is unclear, the phenomenon seems to be an unprecedented type of intercellular trafficking.     

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.     

Distinctions between bovine herpesvirus 1 and herpes simplex virus type 1 VP22 tegument protein subcellular associations

The alphaherpesvirus tegument protein VP22 has been characterized with multiple traits including microtubule reorganization, nuclear localization, and nonclassical intercellular trafficking. However, all these data were derived from studies using herpes simplex virus type 1 (HSV-1) and may not apply to VP22 homologs of other alphaherpesviruses. We compared subcellular attributes of HSV-1 VP22 (HVP22) with bovine herpesvirus 1 (BHV-1) VP22 (BVP22) using green fluorescent protein (GFP)-fused VP22 expression vectors. Fluorescence microscopy of cell lines transfected with these constructs revealed differences as well as similarities between the two VP22 homologs. Compared to that of HVP22, the BVP22 microtubule interaction was much less pronounced. The VP22 nuclear interaction varied, with a marbled or halo appearance for BVP22 and a speckled or nucleolus-bound appearance for HVP22. Both VP22 homologs associated with chromatin at various stages of mitosis and could traffic from expressing cells to the nuclei of nonexpressing cells. However, distinct qualitative differences in microtubule, nuclear, and chromatin association as well as trafficking were observed. The differences in VP22 homolog characteristics revealed in this study will help define VP22 function within HSV-1 and BHV-1 infection.     

Comprehensive characterization of interaction complexes of herpes simplex virus type 1 ICP22, UL3, UL4, and UL20.5

It has been reported that herpes simplex virus type 1 UL3, UL4, and UL20.5 proteins are localized to small, dense nuclear bodies together with ICP22 in infected cells. In the present study, we comprehensively characterized these interactions by subcellular colocalization, coimmunoprecipitation, and bimolecular fluorescence complementation assays. For the first time, it was demonstrated that both UL3 and UL20.5 are targeted to small, dense nuclear bodies by a direct interaction with ICP22, whereas UL4 colocalizes with ICP22 through its interaction with UL3 but not UL20.5 or ICP22. There was no detectable interaction between UL3 and UL20.5.     

Replication of ICP0-null mutant herpes simplex virus type 1 is restricted by both PML and Sp100

Herpes simplex virus type 1 (HSV-1) mutants that fail to express the viral immediate-early protein ICP0 have a pronounced defect in viral gene expression and plaque formation in limited-passage human fibroblasts. ICP0 is a RING finger E3 ubiquitin ligase that induces the degradation of several cellular proteins. PML, the organizer of cellular nuclear substructures known as PML nuclear bodies or ND10, is one of the most notable proteins that is targeted by ICP0. Depletion of PML from human fibroblasts increases ICP0-null mutant HSV-1 gene expression, but not to wild-type levels. In this study, we report that depletion of Sp100, another major ND10 protein, results in a similar increase in ICP0-null mutant gene expression and that simultaneous depletion of both proteins complements the mutant virus to a greater degree. Although chromatin assembly and modification undoubtedly play major roles in the regulation of HSV-1 infection, we found that inhibition of histone deacetylase activity with trichostatin A was unable to complement the defect of ICP0-null mutant HSV-1 in either normal or PML-depleted human fibroblasts. These data lend further weight to the hypothesis that ND10 play an important role in the regulation of HSV-1 gene expression.     

The UL25 gene product of herpes simplex virus type 1 is involved in uncoating of the viral genome

Studies on the herpes simplex virus type 1 UL25-null mutant KUL25NS have shown that the capsid-associated UL25 protein is required at a late stage in the encapsidation of viral DNA. Our previous work on UL25 with the UL25 temperature-sensitive (ts) mutant ts1204 also implicated UL25 in a role at very early times in the virus growth cycle, possibly at the stage of penetration of the host cell. We have reexamined this mutant and discovered that it had an additional ts mutation elsewhere in the genome. The ts1204 UL25 mutation was transferred into wild-type (wt) virus DNA, and the UL25 mutant ts1249 was isolated and characterized to clarify the function of UL25 at the initial stages of virus infection. Indirect immunofluorescence assays and in situ hybridization analysis of virus-infected cells revealed that the mutant ts1249 was not impaired in penetration of the host cell but had an uncoating defect at the nonpermissive temperature. When ts1249-infected cells were incubated initially at the permissive temperature to allow uncoating of the viral genome and subsequently transferred to the restrictive temperature, a DNA-packaging defect was evident. The results suggested that ts1249, like KUL25NS, had a block at a late stage of DNA packaging and that the packaged genome was shorter than the full-length genome. Examination of ts1249 capsids produced at the nonpermissive temperature revealed that, in comparison with wt capsids, they contained reduced amounts of UL25 protein, thereby providing a possible explanation for the failure of ts1249 to package full-length viral DNA.     

Recruitment of polymerase to herpes simplex virus type 1 replication foci in cells expressing mutant primase (UL52) proteins

The ordered assembly of the herpes simplex virus (HSV) type 1 replication apparatus leading to replication compartments likely involves the initial assembly of five viral replication proteins, ICP8, UL9, and the heterotrimeric helicase-primase complex (UL5-UL8-UL52), into replication foci. The polymerase and polymerase accessory protein are subsequently recruited to these foci. Four stages of viral infection (stages I to IV) have been described previously (J. Burkham, D. M. Coen, and S. K. Weller, J. Virol. 72:10100-10107, 1998). Of these, stage III foci are equivalent to the previously described promyelocytic leukemia protein (PML)-associated prereplicative sites and contain all seven replication proteins. We constructed a series of mutations in the putative primase subunit, UL52, of the helicase-primase and have analyzed the mutant proteins for their abilities to form intermediates leading to the formation of replication compartments. The results shown in this paper are consistent with the model that the five proteins, ICP8, UL5, UL8, UL9, and UL52, form a scaffold and that formation of this scaffold does not rely on enzymatic functions of the helicase and primase. Furthermore, we demonstrate that recruitment of polymerase to this scaffold requires the presence of an active primase subunit. These results suggest that polymerase recruitment to replication foci requires primer synthesis. Furthermore, they support the existence of two types of stage III intermediates in the formation of replication compartments: stage IIIa foci, which form the scaffold, and stage IIIb foci, which contain, in addition, HSV polymerase, the polymerase accessory subunit, and cellular factors such as PML.     

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 spread of herpes simplex virus type 1 from trigeminal neurons to the murine cornea: an immunoelectron microscopy study

An animal model has been developed to clarify the mechanism for spread of herpes simplex virus (HSV) from neuron to epithelial cells in herpetic epithelial keratitis. HSV was introduced into the murine trigeminal ganglion via stereotaxic guided injection. After 2 to 5 days, the animals were euthanized. Ganglia and corneas were prepared for light and electron microscopic immunocytochemistry with antisera to HSV. At 2 days, labeled axons were identified in the stromal layer. At 3 days, we could detect immunoreactive profiles of trigeminal ganglion cell axons that contained many vesicular structures. By 3 and 4 days, the infection had spread to all layers of epithelium, and the center of a region of infected epithelium appeared thinned. At 5 day, fewer basal cells appeared infected, although infection persisted in superficial cells where it had expanded laterally. Mature HSV was found in the extracellular space surrounding wing and squamous cells. Viral antigen was expressed in small pits along the apical surfaces of wing and squamous cells but not at the basal surface of these cells or on basal cells. This polarized expression of viral antigen resulted in the spread of HSV to superficial cells and limited lateral spread to neighboring basal cells. The pathogenesis of HSV infection in these mice may serve as a model of the human recurrent epithelial disease in the progression of focal sites of infection and transfer from basal to superficial cells.