William T. Ruyechan

The coactivator host cell factor-1 mediates Set1 and MLL1 H3K4 trimethylation at herpesvirus immediate early promoters for initiation of infection

Originally identified as an essential component of the herpes simplex virus immediate early (IE) gene enhancer complex, the transcriptional coactivator host cell factor-1 (HCF-1) has been implicated in a broad range of cellular regulatory circuits. The protein mediates activation through multiple interactions with transcriptional activators, coactivators, and chromatin remodeling complexes. However, the mechanisms involved in HCF-1-dependent transcriptional stimulation were undefined. By using a minimal HCF-1-dependent promoter and a model activator, the varicella zoster IE62 protein, it was determined that HCF-1 was not required for the assembly of the RNAPII basal complex, which depended solely on IE62 in conjunction with the cellular factor Sp1. In contrast, HCF-1 was required for recruitment of the histone methyltransferases Set1 and MLL1 (mixed-lineage leukemia 1), leading to histone H3K4 trimethylation and transcriptional activation. Similarly, in a varicella zoster virus lytic infection, HCF-1, Set1, and MLL1 were recruited to the viral genomic IE promoter, suggesting an essential role for HCF-1 in chromatin modification and remodeling during initiation of lytic infection. The results indicate that one biological rationale for the incorporation of the viral IE activators in the viral particle is to recruit HCF-1/histone methyltransferase complexes and promote assembly of the viral IE gene promoters into transcriptionally active chromatin. These studies also contribute to the model whereby the induced nuclear transport of HCF-1 in sensory neurons may be critical to the reactivation of latent herpesviruses by promoting the activation of chromatin modifications.

The Varicella-Zoster virus immediate early protein, IE62, can positively regulate its cognate promoter

Varicella-Zoster virus (VZV) is a neurotropic alphaherpes virus closely related to herpes simplex virus (HSV). However, unlike its close relative HSV, VZV lacks a functional alpha-TIF (alpha-gene transinducing factor) that activates the transcription of immediate early genes during the initial events of the virus life cycle. Hence, in the absence of a functional alpha-TIF, the mechanism triggering the expression of immediate early genes in VZV at present remains unclear. Accumulating evidence indicates that the gene product of the putative immediate early gene ORF62 (IE62) plays a pivotal role in activating VZV genes of all three putative kinetic classes, namely immediate early (alpha), early (beta), and late (gamma) classes of VZV genes. In the present study, we show that IE62 can positively autoregulate its cognate promoter using a transient transfection assay, both in lymphocytes and in neural cells. In the same system, we can also demonstrate activation of the VZV IE62 promoter by HSV ICP4. By deletion analysis and oligonucleotide-directed site-specific mutagenesis we have localized specific regions in the IE62 promoter/upstream sequences that mediate inducibility by IE62 and HSV ICP4, and provide evidence that this promoter activation by these two proteins may be through different mechanisms. These data, taken together with the recent demonstration of the presence of IE62 in the VZ virion tegument (Kinchington, P.R., Hoagland, J.K., Arvin, A.M., Ruyechan, W.T., and Hay, J. 1992. J. Virol. 66, 359-366) provides a possible mechanism by which the triggering of VZV gene expression occurs in the absence of a functional alpha-TIF protein.

Varicella-Zoster Virus Immediate-Early Protein 62 Blocks Interferon Regulatory Factor 3 (IRF3) Phosphorylation at Key Serine Residues: a Novel Mechanism of IRF3 Inhibition among Herpesviruses

ABSTRACT Varicella-zoster virus (VZV) is an alphaherpesvirus that is restricted to humans. VZV infection of differentiated cells within the host and establishment of latency likely require evasion of innate immunity and limited secretion of antiviral cytokines. Since interferons (IFNs) severely limit VZV replication, we examined the ability of VZV to modulate the induction of the type I IFN response in primary human embryonic lung fibroblasts (HELF). IFN-β production was not detected, and transcription of two interferon response factor 3 (IRF3)-dependent interferon-stimulated genes (ISGs), ISG54 and ISG56, in response to poly(I:C) stimulation was downregulated in VZV-infected HELF. Inhibition of IRF3 function did not require VZV replication; the viral immediate-early protein 62 (IE62) alone was sufficient to produce this effect. IE62 blocked TBK1-mediated IFN-β secretion and IRF3 function, as shown in an IFN-stimulated response element (ISRE)-luciferase reporter assay. However, IRF3 function was preserved if constitutively active IRF3 (IRF3-5D) was expressed in VZV-infected or IE62-transfected cells, indicating that VZV interferes with IRF3 phosphorylation. IE62-mediated inhibition was mapped to blocking phosphorylation of at least three serine residues on IRF3. However, IE62 binding to TBK1 or IRF3 was not detected and IE62 did not perturb TBK1-IRF3 complex formation. IE62-mediated inhibition of IRF3 function was maintained even if IE62 transactivator activity was disrupted. Thus, IE62 has two critical but discrete roles following VZV entry: to induce expression of VZV genes and to disarm the IFN-dependent antiviral defense through a novel mechanism that prevents IRF3 phosphorylation.

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

ABSTRACT 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 US1.5 protein, which is expressed colinearly with ICP22 (US1). 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.

Complete DNA Sequence Analyses of the First Two Varicella-Zoster Virus Glycoprotein E (D150N) Mutant Viruses Found in North America: Evolution of Genotypes with an Accelerated Cell Spread Phenotype

ABSTRACT Varicella-zoster virus (VZV) is considered to be one of the most genetically stable of all the herpesviruses. Yet two VZV strains with a D150N missense mutation within the gE glycoprotein were isolated in North America in 1998 and 2002. The mutant strains have an accelerated cell spread phenotype, which distinguishes them from all wild-type and laboratory viruses. Since the VZV genome contains 70 additional open reading frames (ORFs), the possibility existed that the phenotypic change was actually due to an as-yet-undiscovered mutation or deletion elsewhere in the genome. To exclude this hypothesis, the entire genomes of the two mutant viruses were sequenced and found to contain 124,883 (VZV-MSP) and 125,459 (VZV-BC) nucleotides. Coding single-nucleotide polymorphisms (SNPs) were identified in 14 ORFs. One missense mutation was discovered in gH, but none was found in gB, gI, gL, or gK. There were no coding SNPs in the major regulatory protein ORF 62. One polymorphism was discovered which could never have been anticipated based on current knowledge of herpesvirus genomics, namely, the origins of replication differed from those in the prototype strain but not in a manner expected to affect cell spread. When the two complete mutant VZV sequences were surveyed in their entirety, the most reasonable conclusion was that the increased cell spread phenotype was dependent substantially or solely on the single D150N polymorphism in glycoprotein gE. The genomic results also expanded the evolutionary database by identifying which VZV ORFs were more likely to mutate over time.

Promoter Sequences of Varicella-Zoster Virus Glycoprotein I Targeted by Cellular Transactivating Factors Sp1 and USF Determine Virulence in Skin and T Cells in SCIDhu Mice In Vivo

ABSTRACT Varicella-zoster virus (VZV) glycoprotein I is dispensable in cell culture but necessary for infection of human skin and T cells in SCIDhu mice in vivo. The gI promoter contains an activating upstream sequence that binds the cellular transactivators specificity factor 1 (Sp1) and upstream stimulatory factor (USF) and an open reading frame 29 (ORF29)-responsive element (29RE), which mediates enhancement by ORF29 DNA binding protein of immediate-early 62 (IE62)-induced transcription. Recombinants, rOKAgI-Sp1 and rOKAgI-USF, with two base pair substitutions in Sp1 or USF sites, replicated like rOKA in vitro, but infectivity of rOKAgI-Sp1 was significantly impaired in skin and T cells in vivo. A double mutant, rOKAgI-Sp1/USF, did not replicate in skin but yielded low titers of infectious virus in T cells. The repaired protein, rOKAgI:rep-Sp1/USF, was as infectious as rOKA. Thus, disrupting gI promoter sites for cellular transactivators altered VZV virulence in vivo, with variable consequences related to the cellular factor and the host cell type. Mutations in the 29RE of the gI promoter were made by substituting each of four 10-bp blocks in this region with a 10-bp sequence, GATAACTACA, that was predicted to interfere with enhancer effects of the ORF29 protein. One of these mutants, which was designated rOKAgI-29RE-3, had diminished replication in skin and T cells, indicating that ORF29 protein-mediated enhancement of gI expression contributes to VZV virulence. Mutations within promoters of viral genes that are nonessential in vitro should allow construction of recombinant herpesviruses that have altered virulence in specific host cells in vivo and may be useful for designing herpesviral gene therapy vectors and attenuated viral vaccines.

Combinatorial transcription of herpes simplex virus and varicella zoster virus immediate early genes is strictly determined by the cellular coactivator HCF-1.

The mammalian transcriptional coactivator host cell factor-1 (HCF-1) functions in concert with Oct-1 and VP16 to assemble the herpes simplex virus (HSV) immediate early (IE) transcription enhancer core complexes that mediate the high level transcription of these genes upon infection. Although this transcriptional model has been well characterized in vitro, the requirements and significance of the components have not been addressed. Oct-1 was previously determined to be critical but not essential for HSV IE gene expression. In contrast, RNA interference-mediated depletion of HCF-1 resulted in abrogation of HSV IE gene expression. The HSV IE gene enhancer domain is a model of combinatorial transcription and consists of the core enhancer and multiple binding sites for factors such as Sp1 and GA-binding protein. It was striking that HCF-1 was strictly required for VP16-mediated transcriptional induction via the core enhancer as well as for basal level transcription mediated by GA-binding protein and Sp1. HCF-1 was also found to be essential for the induction of varicella zoster virus IE gene expression by ORF10, the VZV ortholog of the HSV IE transactivator VP16, and the autostimulatory IE62 protein. The critical dependence upon HCF-1 demonstrates that this cellular component is a key factor for control of HSV and VZV IE gene expression by functioning as the common element for distinct factors cooperating at the IE gene enhancers. The requirements for this protein supports the model whereby the regulated transport of HCF-1 from the cytoplasm to the nucleus in sensory neurons may control IE gene expression and reactivation of these viruses from the latent state.

Memory cytotoxic T cell responses to viral tegument and regulatory proteins encoded by open reading frames 4, 10, 29, and 62 of varicella-zoster virus.

Cytotoxic T cell recognition of tegument and regulatory proteins encoded by open reading frames (ORFs) 4, 10, 29, and 62 of varicella-zoster virus (VZV) was evaluated using limiting dilution conditions to estimate the precursor frequencies of memory T cells specific for these proteins in immune subjects. Responder cell frequencies for ORFs 4, 10, and 62 gene products, which are virion tegument components and function as immediate early viral transactivating proteins, were equivalent. CTLp recognition of VZV proteins made in latently infected cells, which include ORF4 and ORF62 proteins, was not maintained preferentially when compared to ORF10 protein, which has not been shown to be expressed during latency. T cell recognition of ORF29 protein, the major DNA binding protein, which is expressed during replication but not incorporated into the virion tegument, was less common than responses to ORFs 4, 10, and 62 gene products. Older individuals had diminished numbers of memory CTLp that lysed autologous targets expressing IE62 protein; these responses were increased after immunization with live attenuated varicella vaccine to the range observed in younger adults. Adaptive immunity to VZV is characterized by a broad repertoire of memory CTL responses to proteins that comprise the virion tegument and regulate viral gene expression in infected cells.

Interaction between the Varicella Zoster Virus IE62 Major Transactivator and Cellular Transcription Factor Sp1

The varicella zoster virus (VZV) IE62 protein is involved in the activation of expression of all three kinetic classes of VZV proteins. Analysis of the viral promoter for VZV glycoprotein I has shown that the cellular factor Sp1 is involved in or required for the observed IE62 mediated activation. Co-immunoprecipitation experiments show that the two proteins are present in a complex in VZV-infected cells. Protein affinity pull-down assays using recombinant proteins showed that IE62 and Sp1 interact in the absence of any other viral and cellular proteins. Mapping studies using GST-fusion proteins containing truncations of IE62 and Sp1 have delimited the interacting regions to amino acids 612–778 in Sp1 and amino acids 226–299 in IE62. The region identified in Sp1 is involved in DNA-binding, synergistic Sp1 activation, and Sp1 interaction with cellular transcription factors. The interacting region identified in IE62 overlaps with or borders on sites involved in interactions with the VZV IE4 protein and the cellular factors TBP and TFIIB. Assays using wild-type and mutant promoter elements indicate that Sp1 is involved in recruitment of IE62 to the gI promoter and IE62 enhances Sp1 and TBP binding.

Immunity in strain 2 guinea-pigs inoculated with vaccinia virus recombinants expressing varicella-zoster virus glycoproteins I, IV, V or the protein product of the immediate early gene 62

The immunogenicity of specific varicella-zoster virus (VZV) proteins, with emphasis upon cell-mediated immune responses, was evaluated by immunizing strain 2 guinea-pigs with vaccinia virus recombinants that express gpI (vac-gpI), gpIV (vac-gpIV) and gpV (vac-gpV) or the IE-62 protein (vac-IE-62). Vac-gpI elicited the highest initial mean T cell proliferation response [stimulation index (S.I.) 3.8 +/- 0.9 S.E.M.] whereas inoculation with vac-gpV produced the lowest primary T cell response (S.I. 2.5 +/- 1.1 S.E.M.). T cell proliferation was detected for a shorter period after immunization with vac-gpV compared to vac-gpI, vac-gpIV or vac-IE-62. A comparison of the immunogenicity of vac-gpI and vac-IE-62 with the same proteins prepared by immunoaffinity purification showed that immunization with these proteins in either form elicited virus-specific IgG antibodies and T cell recognition. The presence or absence of IgG antibodies to the IE-62 protein was used to assess protection against challenge with guinea-pig cell-adapted infectious VZV in animals that had been inoculated with vac-gpI, vac-gpIV or vac-gpV. Immunization with vac-gpI and vac-gpIV restricted VZV replication but all animals given vac-gpV developed antibodies to IE-62 after challenge with infectious VZV. Priming of the T lymphocyte response was observed in all animals immunized with VZV-vaccinia virus recombinants after subsequent exposure to infectious VZV. These experiments with VZV vac-gpI, vac-gpIV and vac-gpV in guinea-pigs suggest variability in the capacity of herpesviral glycoproteins to elicit cell-mediated immunity in vivo. Induction of virus-specific immunity using IE-62 means that this major tegument protein of VZV could be a useful component for vaccine development.