Functions of the ORF9-to-ORF12 gene cluster in varicella-zoster virus replication and in the pathogenesis of skin infection

The gene cluster composed of varicella-zoster virus (VZV) open reading frame 9 (ORF9) to ORF12 encodes four putative tegument proteins and is highly conserved in most alphaherpesviruses. In these experiments, the genes within this cluster were deleted from the VZV parent Oka (POKA) individually or in combination, and the consequences for VZV replication were evaluated with cultured cells in vitro and with human skin xenografts in SCID mice in vivo. As has been reported for ORF10, ORF11 and ORF12 were dispensable for VZV replication in melanoma and human embryonic fibroblast cells. In contrast, deletion of ORF9 was incompatible with the recovery of infectious virus. ORF9 localized to the virion tegument and formed complexes with glycoprotein E, which is an essential protein, in VZV-infected cells. Recombinants lacking ORF10 and ORF11 (POKADelta10/11), ORF11 and ORF12 (POKADelta11/12), or ORF10, ORF11 and ORF12 (POKADelta10/11/12) were viable in cultured cells. Their growth kinetics did not differ from those of POKA, and nucleocapsid formation and virion assembly were not disrupted. In addition, these deletion mutants showed no differences compared to POKA in infectivity levels for primary human tonsil T cells. Deletion of ORF12 had no effect on skin infection, whereas replication of POKADelta11, POKADelta10/11, and POKADelta11/12 was severely reduced, and no virus was recovered from skin xenografts inoculated with POKADelta10/11/12. These results indicate that with the exception of ORF9, the individual genes within the ORF9-to-ORF12 gene cluster are dispensable and can be deleted simultaneously without any apparent effect on VZV replication in vitro but that the ORF10-to-ORF12 cluster is essential for VZV virulence in skin in vivo.     

The immediate-early 63 protein of Varicella-Zoster virus: analysis of functional domains required for replication in vitro and for T-cell and skin tropism in the SCIDhu model in vivo

The immediate-early 63-kDa (IE63) protein of varicella-zoster virus (VZV) is a phosphoprotein encoded by open reading frame (ORF) ORF63/ORF70. To identify functional domains, 22 ORF63 mutations were evaluated for effects on IE63 binding to the major VZV transactivator, IE62, and on IE63 phosphorylation and nuclear localization in transient transfections, and after insertion into the viral genome with VZV cosmids. The IE62 binding site was mapped to IE63 amino acids 55 to 67, with R59/L60 being critical residues. Alanine substitutions within the IE63 center region showed that S165, S173, and S185 were phosphorylated by cellular kinases. Four mutations that changed two putative nuclear localization signal (NLS) sequences altered IE63 distribution to a cytoplasmic/nuclear pattern. Only three of 22 mutations in ORF63 were compatible with recovery of infectious VZV from our cosmids, but infectivity was restored by inserting intact ORF63 into each mutated cosmid. The viable IE63 mutants had a single alanine substitution, altering T171, S181, or S185. These mutants, rOKA/ORF63rev[T171], rOKA/ORF63rev[S181], and rOKA/ORF63rev[S185], produced less infectious virus and had a decreased plaque phenotype in vitro. ORF47 kinase protein and glycoprotein E (gE) synthesis was reduced, indicating that IE63 contributed to optimal expression of early and late gene products. The three IE63 mutants replicated in skin xenografts in the SCIDhu mouse model, but virulence was markedly attenuated. In contrast, infectivity in T-cell xenografts was not altered. Comparative analysis suggested that IE63 resembled the herpes simplex virus type 1 U(S)1.5 protein, which is expressed colinearly with ICP22 (U(S)1). In summary, most mutations of ORF63 made with our VZV cosmid system were lethal for infectivity. The few IE63 changes that were tolerated resulted in VZV mutants with an impaired capacity to replicate in vitro. However, the IE63 mutants were attenuated in skin but not T cells in vivo, indicating that the contribution of the IE63 tegument/regulatory protein to VZV pathogenesis depends upon the differentiated human cell type which is targeted for infection within the intact tissue microenvironment.     

Mutational analysis of open reading frames 62 and 71, encoding the varicella-zoster virus immediate-early transactivating protein,IE62, and effects on replication in vitro and in skin xenografts in the SCID-hu mouse in vivo

The varicella-zoster virus (VZV) genome has unique long (U(L)) and unique short (U(S)) segments which are flanked by internal repeat (IR) and terminal repeat (TR) sequences. The immediate-early 62 (IE62) protein, encoded by open reading frame 62 (ORF62) and ORF71 in these repeats, is the major VZV transactivating protein. Mutational analyses were done with VZV cosmids generated from parent Oka (pOka), a low-passage clinical isolate, and repair experiments were done with ORF62 from pOka and vaccine Oka (vOka), which is derived from pOka. Transfections using VZV cosmids from which ORF62, ORF71, or the ORF62/71 gene pair was deleted showed that VZV replication required at least one copy of ORF62. The insertion of ORF62 from pOka or vOka into a nonnative site in U(S) allowed VZV replication in cell culture in vitro, although the plaque size and yields of infectious virus were decreased. Targeted mutations in binding sites reported to affect interaction with IE4 protein and a putative ORF9 protein binding site were not lethal. Single deletions of ORF62 or ORF71 from cosmids permitted recovery of infectious virus, but recombination events repaired the defective repeat region in some progeny viruses, as verified by PCR and Southern hybridization. VZV infectivity in skin xenografts in the SCID-hu model required ORF62 expression; mixtures of single-copy recombinant Oka Delta 62 (rOka Delta 62) or rOka Delta 71 and repaired rOka generated by recombination of the single-copy deletion mutants were detected in some skin implants. Although insertion of ORF62 into the nonnative site permitted replication in cell culture, ORF62 expression from its native site was necessary for cell-cell spread in differentiated human skin tissues in vivo.     

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.     

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.     

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.     

Mutational analysis of the repeated open reading frames, ORFs 63 and 70 and ORFs 64 and 69, of varicella-zoster virus

Varicella-zoster virus (VZV) open reading frame 63 (ORF63), located between nucleotides 110581 and 111417 in the internal repeat region, encodes a nuclear phosphoprotein which is homologous to herpes simplex virus type 1 (HSV-1) ICP22 and is duplicated in the terminal repeat region as ORF70 (nucleotides 118480 to 119316). We evaluated the role of ORFs 63 and 70 in VZV replication, using recombinant VZV cosmids and PCR-based mutagenesis to make single and dual deletions of these ORFs. VZV was recovered within 8 to 10 days when cosmids with single deletions were transfected into melanoma cells along with the three intact VZV cosmids. In contrast, VZV was not detected in transfections carried out with a dual deletion cosmid. Infectious virus was recovered when ORF63 was cloned into a nonnative AvrII site in this cosmid, confirming that failure to generate virus was due to the dual ORF63/70 deletion and that replication required at least one gene copy. This requirement may be related to our observation that ORF63 interacts directly with ORF62, the major immediate-early transactivating protein of VZV. ORF64 is located within the inverted repeat region between nucleotides 111565 and 112107; it has some homology to the HSV-1 Us10 gene and is duplicated as ORF69 (nucleotides 117790 to 118332). ORF64 and ORF69 were deleted individually or simultaneously using the VZV cosmid system. Single deletions of ORF64 or ORF69 yielded viral plaques with the same kinetics and morphology as viruses generated with the parental cosmids. The dual deletion of ORF64 and ORF69 was associated with an abnormal plaque phenotype characterized by very large, multinucleated syncytia. Finally, all of the deletion mutants that yielded recombinants retained infectivity for human T cells in vitro and replicated efficiently in human skin in the SCIDhu mouse model of VZV pathogenesis.     

ORF66 protein kinase function is required for T-cell tropism of varicella-zoster virus in vivo

Several functions have been attributed to the serine/threonine protein kinase encoded by open reading frame 66 (ORF66) of varicella-zoster virus (VZV), including modulation of the apoptosis and interferon pathways, down-regulation of major histocompatibility complex class I cell surface expression, and regulation of IE62 localization. The amino acid sequence of the ORF66 protein contains a recognizable conserved kinase domain. Point mutations were introduced into conserved protein kinase motifs to evaluate their importance to ORF66 protein functions. Two substitution mutants were generated, including a G102A substitution, which blocked autophosphorylation and altered IE62 localization, and an S250P substitution, which had no effect on either autophosphorylation or IE62 localization. Both kinase domain mutants grew to titers equivalent to recombinant parent Oka (pOka) in vitro. pOka66G102A had slightly reduced growth in skin, which was comparable to the reduction observed when ORF66 translation was prevented by stop codon insertions in pOka66S. In contrast, infection of T-cell xenografts with pOka66G102A was associated with a significant decrease in infectious virus production equivalent to the impaired T-cell tropism found with pOka66S infection of T-cell xenografts in vivo. Disrupting kinase activity with the G102A mutation did not alter IE62 cytoplasmic localization in VZV-infected T cells, suggesting that decreased T-cell tropism is due to other ORF66 protein functions. The G102A mutation reduced the antiapoptotic effects of VZV infection of T cells. These experiments indicate that the T-cell tropism of VZV depends upon intact ORF66 protein kinase function.