Mutational analysis of varicella-zoster virus major immediate-early protein IE62.

The varicella-zoster virus (VZV) open reading frame 62 encodes an immediate-early protein (IE62) that transactivates expression of various VZV promoters and autoregulates its own expression in transient expression assays. In Vero cells, IE62 was shown to transactivate the expression of all putative immediate-early (IE) and early (E) genes of VZV with an up-regulating effect at low intracellular concentrations. To define the functional domains involved in the regulatory properties of IE62, a large number of in-frame insertions and deletions were introduced into a plasmid-borne copy of the gene encoding IE62. Studies of the regulatory activities of the resultant mutant polypeptides in transient expression assays allowed to delineate protein regions important for repression of its own promoter and for transactivation of a VZV putative immediate-early gene (ORF61) promoter and an early gene (ORF29) promoter. This mutational analysis resulted in the identification of a new functional domain situated at the border between regions 4 and 5 which plays a crucial role in the IE62 regulatory functions. This domain turned out to be very well conserved amongst homologous alphaherpesvirus regulatory proteins and appeared to be rich in bulky hydrophobic and proline residues, similar to the proline-rich region of the CAAT box binding protein CTF-1. By immunofluorescence, a nuclear localization signal has been mapped in region 3.     

Varicella-zoster virus open reading frame 61 protein is functionally homologous to herpes simplex virus type 1 ICP0.

The varicella-zoster virus (VZV) open reading frame 61 (ORF61) protein is thought to be the homolog of herpes simplex virus type 1 (HSV-1) ICP0, based on gene location and limited amino acid homology. However, HSV-1 ICP0 trans activates HSV-1 genes, while VZV ORF61 protein trans represses the function of VZV trans activators on VZV promoters in transient expression assays. To investigate the functional relatedness of HSV-1 ICP0 and VZV ORF61 protein, we established Vero and MeWo cell lines which stably express VZV ORF61 under the control of a metallothionein promoter and performed complementation studies with an HSV-1 ICP0 deletion mutant (7134). Mutant 7134 is impaired for plaque formation and replication at a low multiplicity of infection in cell culture, but these defects were complemented by up to 200-fold in Vero cell lines expressing VZV ORF61. Likewise, the efficiency of plaque formation was improved by up to 100-fold in MeWo cell lines expressing VZV ORF61. A cell line expressing another VZV immediate-early gene product (ORF62) was unable to complement mutant 7134. HSV-1 mutants which are deleted for other HSV-1 immediate-early gene products (ICP4, ICP27) were unable to grow in VZV ORF61-expressing cell lines. These results indicate that, despite marked differences in their sequences and in effects on their cognate promoters in transient expression assays, VZV ORF61 protein is the functional homolog of HSV-1 ICP0.     

Varicella-zoster virus (VZV) open reading frame 61 protein transactivates VZV gene promoters and enhances the infectivity of VZV DNA.

The varicella-zoster virus (VZV) open reading frame 61 (ORF61) protein is the homolog of herpes simplex virus type 1 (HSV-1) ICP0. Both genes are located in similar parts of the genome, their predicted products share a cysteine-rich motif, and cell lines expressing VZV ORF61 are able to complement an HSV-1 ICP0 deletion mutant (H. Moriuchi, M. Moriuchi, H. A. Smith, S. E. Straus, and J. I. Cohen, J. Virol. 66:7303-7308, 1992). In transient expression assays, HSV-1 ICP0 is a transactivator alone and transactivates in synergy with another viral transactivator, ICP4. However, VZV ORF61 represses the activation by VZV-encoded proteins ORF62 (the homolog of ICP4) and ORF4. To further characterize the function of VZV ORF61 and its role(s) in regulation of viral gene expression, we performed transient expression assays using target promoters from VZV, HSV-1, and unrelated viruses. In the absence of other viral activators, VZV ORF61 transactivated most promoters tested. In addition, a cell line stably expressing VZV ORF61 complemented the HSV-1 mutant in 1814, which lacks the transactivating function of VP16. The cell line expressing VZV ORF61 enhanced the infectivity of HSV-1 virion DNA. Moreover, transient expression of VZV ORF61 also enhanced the infectivity of VZV DNA. These results indicate that VZV ORF61 can stimulate expression of HSV-1 and VZV genes at an early stage in the viral replicative cycle and that ORF61 has an important role in VZV gene regulation.     

Phosphorylation of varicella-zoster virus open reading frame (ORF) 62 regulatory product by viral ORF 47-associated protein kinase.

Varicella-zoster virus (VZV) encodes within its unique long region a gene product with protein kinase motifs. In a previous study, we demonstrated that immunoprecipitated VZV open reading frame (ORF) 47 protein was associated with a functional protein kinase activity, on the basis of its ability to both autophosphorylate and phosphorylate artificial substrates. To further define potential substrates of ORF 47-associated protein kinase, we analyzed individual viral phosphoproteins to determine whether any were modified by the viral protein kinase. These candidates included gene products of VZV ORFs 4, 61, 62, and 63, which are homologs of herpes simplex virus type 1 (HSV-1) immediate-early proteins. Each of the above VZV proteins was coimmunoprecipitated with ORF 47 kinase, and the immune complex was incubated in a protein kinase assay. Under these conditions, only the VZV immediate-early ORF 62 protein was phosphorylated by ORF 47-associated protein kinase. The specificity of this phosphorylation event was analyzed by a competition assay in which a recombinant ORF 47 protein lacking enzymatic activity was able to reduce the amount of phosphorylation of ORF 62 protein by VZV ORF 47-associated kinase. To provide an additional evaluation of specificity, the experiment was repeated with [32P]GTP instead of [32P]ATP, because the VZV ORF 47 kinase has the distinctive property of using GTP as a phosphate donor. Again the ORF 62 substrate was phosphorylated. In summary, the VZV ORF 47-associated protein kinase (the HSV-1 UL13 homolog) catalyzed the in vitro phosphorylation of the VZV ORF 62 protein, the homolog of the HSV-1 ICP4 regulatory protein.     

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.     

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.     

Varicella-zoster virus open reading frame 66 protein kinase is required for efficient viral growth in primary human corneal stromal fibroblast cells

Varicella-zoster virus (VZV) open reading frame 66 (ORF66) encodes a serine/threonine protein kinase that is not required for VZV growth in most cell types but is needed for efficient growth in T cells. The ORF66 kinase affects nuclear import and virion packaging of IE62, the major regulatory protein, and is known to regulate apoptosis in T cells. Here, we further examined the importance of ORF66 using VZV recombinants expressing green fluorescent protein (GFP)-tagged functional and kinase-negative ORF66 proteins. VZV virions with truncated or kinase-inactivated ORF66 protein were marginally reduced for growth and progeny yields in MRC-5 fibroblasts but were severely growth and replication impaired in low-passage primary human corneal stromal fibroblasts (PCF). To determine if the growth impairment was due to ORF66 kinase regulation of IE62 nuclear import, recombinant VZVs that expressed IE62 with alanine residues at S686, the suspected target by which ORF66 kinase blocks IE62 nuclear import, were made. IE62 S686A expressed by the VZV recombinant remained nuclear throughout infection and was not packaged into virions. However, the mutant virus still replicated efficiently in PCF cells. We also show that inactivation of the ORF66 kinase resulted in only marginally increased levels of apoptosis in PCF cells, which could not fully account for the cell-specific growth requirement of ORF66 kinase. Thus, the unique short region VZV kinase has important cell-type-specific functions that are separate from those affecting IE62 and apoptosis.     

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.     

Immunization with the immediate-early tegument protein (open reading frame 62) of varicella-zoster virus protects guinea pigs against virus challenge.

The IE62 protein, the primary regulatory protein of varicella-zoster virus (VZV) and the major component of the virion tegument, was an effective immunogen in the guinea pig model of VZV infection, whereas the ORF 29 gene product, a nonstructural DNA replication protein, did not elicit protection. All animals immunized with the ORF 29 protein had cell-associated viremia compared with 2 of 11 guinea pigs given the IE62 protein (P = 0.005). VZV was detected in ganglia from 38% of the animals given the ORF 29 protein and 44% of the control animals compared with 9% of the animals immunized with the IE62 protein (P = 0.04). In contrast to the IE62 protein, immunization with the ORF 29 protein did not prime the animals for an enhanced T-cell response upon challenge with infectious virus. The VZV IE62 protein has potential value as a vaccine component.     

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 immediate-early protein IE62 is a major component of virus particles.

Varicella-zoster virus (VZV) open reading frame (ORF) 62 potentially encodes a protein with considerable amino acid homology to the herpes simplex virus (HSV) immediate-early regulatory polypeptide ICP4 (or IE3). To identify and characterize its protein product(s) (IE62), we used a rabbit antiserum prepared against a synthetic peptide corresponding to the C-terminal 13 amino acids of the predicted protein. This antiserum reacted with phosphorylated polypeptides of 175 to 180 kDa that were made in VZV-infected cells and in cells infected with a vaccinia virus recombinant expressing IE62, but not in control-infected cells, confirming its specificity and reactivity to the IE62 protein. The antiserum recognized a 175-kDa polypeptide in purified virions that comigrated with a major structural protein. Comparison of this reactivity with that of an antipeptide antiserum directed against the VZV ORF 10 product (homologous to the HSV major structural protein VP16) indicates similar levels of ORF 62 and ORF 10 polypeptides in VZV virions. In contrast, antipeptide antiserum directed against the VZV ORF 29 product, the homolog of the HSV major DNA-binding protein, failed to recognize any protein in our virion preparations. Treatment of virions with detergents that disrupt the virion envelope did not dissociate IE62 from the nucleocapsid-tegument structure of the virion. Differential sensitivity of VZV virion IE62 to trypsin digestion in the presence or absence of Triton X-100 indicates that IE62 is protected from trypsin degradation by the virus envelope; since it is not a nucleocapsid protein, we conclude that it is part of the tegument. Finally, we show that the virion 175-kDa protein either can autophosphorylate or is a major substrate in vitro for virion-associated protein kinase activity.     

Infection of human T lymphocytes with varicella-zoster virus: an analysis with viral mutants and clinical isolates

Varicella-zoster virus (VZV) disseminates in the body in peripheral blood mononuclear cells during chickenpox. Up to 1 in 10,000 mononuclear cells are infected during the viremic phase of the disease. We developed an in vitro system to infect human mononuclear cells with VZV by using umbilical cord blood. In this system, 3 to 4% of T cells were infected with VZV. VZV mutants unable to express certain genes, such as open reading frame 47 (ORF47) or ORF66, were impaired for growth in T cells, while other mutants showed little difference from parental virus. VZV unable to express ORF47 was even more impaired for spread from umbilical cord blood cells to melanoma cells in vitro. Early-passage clinical isolates of VZV infected T cells at a similar rate to the Oka vaccine strain; however, the clinical isolates were more efficient in spreading from infected T cells to melanoma cells. This in vitro system for infecting human T cells with VZV should be useful for identifying cellular and viral proteins that are important for virus replication in T cells and for the spread of virus from T cells to other cells.