Varicella-Zoster Virus (VZV) ORF32 Encodes a Phosphoprotein That Is Posttranslationally Modified by the VZV ORF47 Protein Kinase

Varicella-zoster virus (VZV) encodes five gene products that do not have homologs in herpes simplex virus. One of these genes, VZV open reading frame 32 (ORF32), is predicted to encode a protein of 16 kDa. VZV ORF32 protein was shown to be phosphorylated and located in the cytosol of virus-infected cells. Antibody to ORF32 protein immunoprecipitated 16- and 18-kDa phosphoproteins from VZV-infected cells. Since VZV encodes two protein kinases that might phosphorylate ORF32 protein, immunoprecipitations were performed with cells infected with VZV mutants unable to express either of the viral protein kinases. Cells infected with VZV unable to express the ORF66 protein kinase contained both the 16- and 18-kDa ORF32 phosphoproteins; however, cells infected with the VZV ORF47 protein kinase mutant showed only the 16-kDa ORF32 phosphoprotein. Treatment of [³⁵S]methionine-labeled proteins with calf intestine alkaline phosphatase resulted in a decrease in size of the ORF32 proteins from 16 and 18 kDa to 15 and 17 kDa, respectively. VZV unable to express ORF32 protein replicated in human melanoma cells to titers similar to those seen with parental virus; however, VZV unable to express ORF32 was impaired for replication in U20S osteosarcoma cells. Thus, VZV ORF32 protein is posttranslationally modified by the ORF47 protein kinase. Since the VZV ORF47 protein kinase has recently been shown to be critical for replication in human fetal skin and lymphocytes, its ability to modify the ORF32 protein suggests that the latter protein may have a role for VZV replication in human tissues.     

Differential requirement for cell fusion and virion formation in the pathogenesis of varicella-zoster virus infection in skin and T cells

The protein product of varicella-zoster virus (VZV) ORF47 is a serine/threonine protein kinase and tegument component. Evaluation of two recombinants of the Oka strain, rOka47DeltaC, with a C-terminal truncation of ORF47, and rOka47D-N, with a point mutation in the conserved kinase motif, showed that ORF47 kinase function was necessary for optimal VZV replication in human skin xenografts in SCID mice but not in cultured cells. We now demonstrate that rOka47DeltaC and rOka47D-N mutants do not infect human T-cell xenografts. Differences in the growth of kinase-defective ORF47 mutants allowed an examination of requirements for VZV pathogenesis in skin and T cells in vivo. Although virion assembly was reduced and no virion transport to cell surfaces was observed, epidermal cell fusion persisted, and VZV polykaryocytes were generated by rOka47DeltaC and rOka47D-N in skin. Virion assembly was also impaired in vitro, but VZV-induced cell fusion continued to cause syncytia in cultured cells infected with rOka47DeltaC or rOka47D-N. Intracellular trafficking of envelope glycoprotein E and the ORF47 and IE62 proteins, components of the tegument, was aberrant without ORF47 kinase activity. In summary, normal VZV virion assembly appears to require ORF47 kinase function. Cell fusion was induced by ORF47 mutants in skin, and cell-cell spread occurred even though virion formation was deficient. VZV-infected T cells do not undergo cell fusion, and impaired virion assembly by ORF47 mutants was associated with a complete elimination of T-cell infectivity. These observations suggest a differential requirement for cell fusion and virion formation in the pathogenesis of VZV infection in skin and T cells.     

The varicella-zoster virus ORF66 protein induces kinase activity and is dispensable for viral replication.

Varicella-zoster virus (VZV) open reading frames (ORFs) 47 and 66 encode proteins that are homologous to a family of eukaryotic serine-threonine kinases. Prior studies showed that the VZV ORF47 protein has kinase activity in vitro and is dispensable for replication in cultured cells. To examine the role of the ORF66 protein during infection, we constructed VZV recombinants that are unable to express either the ORF66 protein (ROka 66S) or both the ORF47 and ORF66 proteins (ROka 47S/66S). VZV unable to express ORF66 grew to titers similar to those of the parental VZV (ROka) in vitro; however, VZV lacking both ORF66 and ORF47 grew to titers lower than those of ROka. Nuclear extracts from ROka 66S- or ROka 47S-infected cells showed a 48-kDa phosphoprotein(s); a phosphoprotein with a similar size was not present in nuclear extracts from ROka 47S/66S-infected cells. To determine the role of the ORF66 protein in the phosphorylation of specific VZV-encoded proteins, we immunoprecipitated known VZV phosphoproteins (ORF4, ORF62, ORF63, and ORF68 proteins) from nuclear extracts of phosphate-labeled cells infected with ROka, ROka 66S, or ROka 47S/66S. Each of the VZV phosphoproteins was phosphorylated to a similar extent in the presence or absence of either the ORF66 protein or both the ORF66 and ORF47 proteins. From these studies we conclude (i) neither ORF66 alone nor ORF66 and ORF47 in combination are essential for VZV growth in cultured cells, (ii) ORF66 either is a protein kinase or induces protein kinase activity during infection, and (iii) the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 do not require either ORF66 alone or ORF66 and ORF47 for phosphorylation in vitro.     

Phosphorylation by the varicella-zoster virus ORF47 protein serine kinase determines whether endocytosed viral gE traffics to the trans-Golgi network or recycles to the cell membrane

Like all alphaherpesviruses, varicella-zoster virus (VZV) infection proceeds by both cell-cell spread and virion production. Virions are enveloped within vacuoles located near the trans-Golgi network (TGN), while in cell-cell spread, surface glycoproteins fuse cells into syncytia. In this report, we delineate a potential role for serine/threonine phosphorylation of the cytoplasmic tail of the predominant VZV glycoprotein, gE, in these processes. The fact that VZV gE (formerly called gpI) is phosphorylated has been documented (E. A. Montalvo and C. Grose, Proc. Natl. Acad. Sci. USA 83:8967-8971, 1986), although respective roles of viral and cellular protein kinases have never been delineated. VZV ORF47 is a viral serine protein kinase that recognized a consensus sequence similar to that of casein kinase II (CKII). During open reading frame 47 (ORF47)-specific in vitro kinase assays, ORF47 phosphorylated four residues in the cytoplasmic tail of VZV gE (S593, S595, T596, and T598), thus modifying the known phosphofurin acidic cluster sorting protein 1 domain. CKII phosphorylated gE predominantly on the two threonine residues. In wild-type-virus-infected cells, where ORF47-mediated phosphorylation predominated, gE endocytosed and relocalized to the TGN. In cells infected with a VZV ORF47-null mutant, internalized VZV gE recycled to the plasma membrane and did not localize to the TGN. The mutant virus also formed larger syncytia than the wild-type virus, linking CKII-mediated gE phosphorylation with increased cell-cell spread. Thus, ORF47 and CKII behaved as "team players" in the phosphorylation of VZV gE. Taken together, the results showed that phosphorylation of VZV gE by ORF47 or CKII determined whether VZV infection proceeded toward a pathway likely involved with either virion production or cell-cell spread.     

T-cell tropism and the role of ORF66 protein in pathogenesis of varicella-zoster virus infection

The pathogenesis of varicella-zoster virus (VZV) involves a cell-associated viremia during which infectious virus is carried from sites of respiratory mucosal inoculation to the skin. We now demonstrate that VZV infection of T cells is associated with robust virion production and modulation of the apoptosis and interferon pathways within these cells. The VZV serine/threonine protein kinase encoded by ORF66 is essential for the efficient replication of VZV in T cells. Preventing ORF66 protein expression by stop codon insertion (pOka66S) impaired the growth of the parent Oka (pOka) strain in T cells in SCID-hu T-cell xenografts in vivo and reduced formation of VZV virions. The lack of ORF66 protein also increased the susceptibility of infected T cells to apoptosis and reduced the capacity of the virus to interfere with induction of the interferon (IFN) signaling pathway following exposure to IFN-gamma. However, preventing ORF66 protein expression only slightly reduced growth in melanoma cells in culture and did not diminish virion formation in these cells. The pOka66S virus showed only a slight defect in growth in SCID-hu skin implants compared with intact pOka. These observations suggest that the ORF66 kinase plays a unique role during infection of T cells and supports VZV T-cell tropism by contributing to immune evasion and enhancing survival of infected T cells.     

IL-12, IFN-gamma, and TNF-alpha released from mononuclear cells inhibit the spread of varicella-zoster virus at an early stage of varicella

The activity of mononuclear cells to inhibit plaque formation of varicella-zoster virus (VZV) was investigated by an in vitro infectious center assay. Peripheral blood mononuclear cells (PBMC) inhibited VZV plaque formation by co-cultivation with VZV-infected fibroblasts. As compared to mononuclear cells from normal individuals, mononuclear cells from umbilical cord blood and from patients receiving corticosteroids showed a significant decrease in the ability to inhibit viral replication. This ability was significantly increased for mononuclear cells collected during the acute phase of varicella. PBMC obtained from patients in the acute phase of varicella produced significantly higher amounts of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-12 in the supernatant compared with those of healthy individuals. These data suggest that the cytokines have an important role in the inhibition of the spread of VZV at an early stage of varicella. Th1 type adaptive immunity might play a major role in VZV infection.     

Deletion of the varicella-zoster virus large subunit of ribonucleotide reductase impairs growth of virus in vitro.

Cells infected with varicella-zoster virus (VZV) express a viral ribonucleotide reductase which is distinct from that present in uninfected cells. VZV open reading frames 18 and 19 (ORF18 and ORF19) are homologous to the herpes simplex virus type 1 genes encoding the small and large subunits of ribonucleotide reductase, respectively. We generated recombinant VZV by transfecting cultured cells with four overlapping cosmid DNAs. To construct a virus lacking ribonucleotide reductase, we deleted 97% of VZV ORF19 from one of the cosmids. Transfection of this cosmid with the other parental cosmids yielded a VZV mutant with a 2.3-kbp deletion confirmed by Southern blot analysis. Virus-specific ribonucleotide reductase activity was not detected in cells infected with VZV lacking ORF19. Infection of melanoma cells with ORF19-deleted VZV resulted in plaques smaller than those produced by infection with the parental VZV. The mutant virus also exhibited a growth rate slightly slower than that of the parental virus. Chemical inhibition of the VZV ribonucleotide reductase has been shown to potentiate the anti-VZV activity of acyclovir. Similarly, the concentration of acyclovir required to inhibit plaque formation by 50% was threefold lower for the VZV ribonucleotide reductase deletion mutants than for parental virus. We conclude that the VZV ribonucleotide reductase large subunit is not essential for virus infection in vitro; however, deletion of the gene impairs the growth of VZV in cell culture and renders the virus more susceptible to inhibition by acyclovir.     

The varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase is dispensable for viral replication and is not required for phosphorylation of ORF63 protein, the VZV homolog of herpes simplex virus ICP22.

To investigate the role of varicella-zoster virus (VZV) open reading frame 47 (ORF47) protein kinase during infection, a VZV mutant was generated in which two contiguous stop codons were introduced into ORF47, thus eliminating expression of the ORF47 kinase. ORF47 kinase was not essential for the growth of VZV in cultured cells, and the growth rate of the VZV mutant lacking ORF47 protein was indistinguishable from that of parental VZV. Nuclear extracts from cells infected with parental VZV contained several phosphorylated proteins which were not detected in extracts from cells infected with the ORF47 mutant. The herpes simplex virus type 1 (HSV-1) UL13 protein (the homolog of VZV ORF47 protein) is responsible for the posttranslational processing associated with phosphorylation of HSV-1 ICP22 (the homolog of VZV ORF63 protein). Immunoprecipitation of 32P-labeled proteins from cells infected with parental virus and those infected with ORF47 mutant virus yielded similar amounts of the VZV phosphoproteins encoded by ORF4, ORF62, ORF63, and ORF68 (VZV gE), and the electrophoretic migration of these proteins was not affected by the lack of ORF47 kinase. Therefore, while the VZV ORF47 protein is capable of phosphorylating several cellular or viral proteins, it is not required for phosphorylation of the ORF63 protein in virus-infected cells.     

Deletion in open reading frame 49 of varicella-zoster virus reduces virus growth in human malignant melanoma cells but not in human embryonic fibroblasts

The ORF49 gene product (ORF49p) of the varicella-zoster virus (VZV) is likely a myristylated tegument protein, and its homologs are conserved across the herpesvirus subfamilies. The UL11 gene of herpes simplex virus type 1 and of pseudorabies virus and the UL99 gene of human cytomegalovirus are the homologs of ORF49 and have been well characterized by using mutant viruses; however, little research on the VZV ORF49 gene has been reported. Here we report on VZV ORF49p expression, subcellular localization, and effect on viral spread in vitro. ORF49p was expressed during the late phase of infection and located in the juxtanuclear region of the cytoplasm, where it colocalized mainly with the trans-Golgi network-associated protein. ORF49p was incorporated into virions and showed a molecular mass of 13 kDa in VZV-infected cells and virions. To elucidate the role of the ORF49 gene, we constructed a mutant virus that lacked a functional ORF49. No differences in plaque size or cell-cell spread were observed in human embryonic fibroblast cells, MRC-5 cells, infected with the wild-type or the mutant virus. However, the mutant virus showed diminished cell-cell infection in a human malignant melanoma cell line, MeWo cells. Therefore, VZV ORF49p is important for virus growth in MeWo cells, but not in MRC-5 cells. VZV may use different mechanisms for virus growth in MeWo and MRC-5 cells. If so, understanding the role of ORF49p should help elucidate how VZV accomplishes cell-cell infections in different cell types.     

The varicella-zoster virus ORF47 kinase interferes with host innate immune response by inhibiting the activation of IRF3

The innate immune response constitutes the first line of host defence that limits viral spread and plays an important role in the activation of adaptive immune response. Viral components are recognized by specific host pathogen recognition receptors triggering the activation of IRF3. IRF3, along with NF-κB, is a key regulator of IFN-β expression. Until now, the role of IRF3 in the activation of the innate immune response during Varicella-Zoster Virus (VZV) infection has been poorly studied. In this work, we demonstrated for the first time that VZV rapidly induces an atypical phosphorylation of IRF3 that is inhibitory since it prevents subsequent IRF3 homodimerization and induction of target genes. Using a mutant virus unable to express the viral kinase ORF47p, we demonstrated that (i) IRF3 slower-migrating form disappears; (ii) IRF3 is phosphorylated on serine 396 again and recovers the ability to form homodimers; (iii) amounts of IRF3 target genes such as IFN-β and ISG15 mRNA are greater than in cells infected with the wild-type virus; and (iv) IRF3 physically interacts with ORF47p. These data led us to hypothesize that the viral kinase ORF47p is involved in the atypical phosphorylation of IRF3 during VZV infection, which prevents its homodimerization and subsequent induction of target genes such as IFN-β and ISG15.     

Role of the varicella-zoster virus gene product encoded by open reading frame 35 in viral replication in vitro and in differentiated human skin and T cells in vivo

Although genes related to varicella-zoster virus (VZV) open reading frame 35 (ORF35) are conserved in the herpesviruses, information about their contributions to viral replication and pathogenesis is limited. Using a VZV cosmid system, we deleted ORF35 to produce two null mutants, designated rOkaDelta35(#1) and rOkaDelta35(#2), and replaced ORF35 at a nonnative site, generating two rOkaDelta35/35@Avr mutants. ORF35 Flag-tagged recombinants were made by inserting ORF35-Flag at the nonnative Avr site as the only copy of ORF35, yielding rOkaDelta35/35Flag@Avr, or as a second copy, yielding rOka35Flag@Avr. Replication of rOkaDelta35 viruses was diminished in melanoma and Vero cells in a 6-day analysis of growth kinetics. Plaque sizes of rOkaDelta35 mutants were significantly smaller than those of rOka in melanoma cells. Infection of melanoma cells with rOkaDelta35 mutants was associated with disrupted cell fusion and polykaryocyte formation. The small plaque phenotype was not corrected by growth of rOkaDelta35 mutants in melanoma cells expressing the major VZV glycoprotein E, gE. The rOkaDelta35/35@Avr viruses displayed growth kinetics and plaque morphologies that were indistinguishable from those of rOka. Analysis with ORF35-Flag recombinants showed that the ORF35 gene product localized predominantly to the nuclei of infected cells. Evaluations in the SCIDhu mouse model demonstrated that ORF35 was required for efficient VZV infection of skin and T-cell xenografts, although the decrease in infectivity was most significant in skin. These mutagenesis experiments indicated that ORF35 was dispensable for VZV replication, but deleting ORF35 diminished growth in cultured cells and was associated with attenuated VZV infection of differentiated human skin and T cells in vivo.     

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.     

Differentiation of varicella-zoster virus ORF47 protein kinase and IE62 protein binding domains and their contributions to replication in human skin xenografts in the SCID-hu mouse

To investigate the role of the ORF47 protein kinase of varicella-zoster virus (VZV), we constructed VZV recombinants with targeted mutations in conserved motifs of ORF47 and a truncated ORF47 and characterized these mutants for replication, phosphorylation, and protein-protein interactions in vitro and for infectivity in human skin xenografts in the SCID-hu mouse model in vivo. Previous experiments showed that ROka47S, a null mutant that makes no ORF47 protein, did not replicate in skin in vivo (J. F. Moffat, L. Zerboni, M. H. Sommer, T. C. Heineman, J. I. Cohen, H. Kaneshima, and A. M. Arvin, Proc. Natl. Acad. Sci. USA 95:11969-11974, 1998). The construction of VZV recombinants with targeted ORF47 mutations made it possible to assess the effects on VZV infection of human skin xenografts of selectively abolishing ORF47 protein kinase activity. ORF47 mutations that resulted in a C-terminal truncation or disrupted the DYS kinase motif eliminated ORF47 kinase activity and were associated with extensive nuclear retention of ORF47 and IE62 proteins in vitro. Disrupting ORF47 kinase function also resulted in a marked decrease in VZV replication and cutaneous lesion formation in skin xenografts in vivo. However, infectivity in vivo was not blocked completely as long as the capacity of ORF47 protein to bind IE62 protein was preserved, a function that we identified and mapped to the N-terminal domain of ORF47 protein. These experiments indicate that ORF47 kinase activity is of critical importance for VZV infection and cell-cell spread in human skin in vivo but suggest that it is the formation of complexes between ORF47 and IE62 proteins, both VZV tegument components, that constitutes the essential contribution of ORF47 protein to VZV replication in vivo.     

Varicella-zoster virus glycoprotein I is essential for growth of virus in Vero cells.

Varicella-zoster virus (VZV) encodes at least six glycoproteins. Glycoprotein I (gI), the product of open reading frame 67, is a 58- to 62-kDa glycoprotein found in VZV-infected cells. We constructed two VZV gI deletion mutants. Immunoprecipitation of VZV gE from infected cells indicated that cells infected with VZV deleted for gI expressed a gE that was larger (100 kDa) than that expressed in cells infected with the parental virus (98 kDa). Cell-associated or cell-free VZV deleted for gI grew to lower titers in melanoma cells than did parental VZV. While VZV deleted for gI replicated in other human cells, the mutant virus replicated to very low titers in primary guinea pig and monkey cells and did not replicate in Vero cells. When compared with the parental virus, rescued viruses, in which the gI deletion was restored with a wild-type allele, showed a similarly sized gE and comparable growth patterns in melanoma and Vero cells. VZV deleted for gI entered Vero cells; however, viral DNA synthesis was impaired in these cells. The VZV gI mutant was slightly impaired for adsorption to human cells. Thus, VZV gI is required for replication of the virus in Vero cells, for efficient replication of the virus in nonhuman cells, and for normal processing of gE.