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Abstract: Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella and herpes zoster. In these experiments, cDNA corresponding to 69 VZV open reading frames was added to 42K human cDNA microarrays and used to examine viral as well as cellular gene transcription concurrently in fibroblasts infected with two genetically distinct small plaque VZV mutants, rOka/ORF63rev[T171] and rOkaΔgI. rOka/ORF63rev[T171] has a point mutation in ORF63, which encodes the immediate early regulatory protein, IE63, and rOkaΔgI has a deletion of ORF67, encoding glycoprotein I (gI). rOka/ORF63rev[T171] was deficient in the transcription of several viral genes compared to the recombinant rOka control virus. Deletion of ORF67 had minimal effects on viral gene transcription. Effects of rOka/ORF63rev[T171] and rOkaΔgI on host cell gene transcription were similar to the rOka control, but a few host cell genes were regulated differently in rOkaΔgI-infected cells. Infection of fibroblasts with intact or small plaque VZV mutants was associated with down-regulation of NF-κB and interferon responsive genes, down-regulation of TGF-β responsive genes accompanied by reduced amounts of fibrotic/wound healing response genes (e.g. collagens, follistatin) and activation of cellular proliferation genes, and alteration of neuronal growth markers, as well as cellular genes encoding proteins important in protein and vesicle trafficking. These observations suggest that replication of VZV small plaque mutant viruses and intact VZV have similar consequences for host cell gene transcription in infected cells, and that the small plaque phenotype in these mutants reflects deficiencies in viral gene expression. [Copyright &y& Elsevier]

During primary infection, varicella-zoster virus (VZV) is spread via lymphocytes to skin, where it induces a rash and establishes latency in sensory ganglia. A live, attenuated varicella vaccine (vOka) was generated by using the VZV Oka strain (pOka), but the molecular basis for vOka attenuation remains unknown. Little is known concerning the effects of wild-type or attenuated VZV on cellular gene regulation in the host cells that are critical for pathogenesis. In this study, transcriptional profiles of primary human T cells and fibroblasts infected with VZV in cell culture were determined by using 40,000-spot human eDNA microarrays. Cellular gene transcription in human skin xenografts in SCID mice that were infected with VZV in vivo was also evaluated. The profiles of cellular gene transcripts that were induced or inhibited in infected human foreskin fibroblasts (HFFs), T cells, and skin in response to pOka and vOka infection were similar. However, significant alterations in cellular gene regulation were observed among the three differentiated human cell types that were examined, suggesting specific differences in the biological consequences of VZV infection related to the target cell. Changes in cellular gene transcription detected by microarray analysis were confirmed for selected genes by quantitative real-time reverse transcription-PCR analysis of VZV-infected cells. Interestingly, the transcription of caspase 8 was found to be decreased in infected T cells but not in HFFs or skin, which may signify a tissue-specific antiapoptosis mechanism. The use of microarrays to demonstrate differences in effects on host cell genes in primary, biologically relevant cell types provides background information for experiments to link these various response phenotypes with mechanisms of VZV pathogenesis that are important for the natural course of human infection. [ABSTRACT FROM AUTHOR]

Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella and herpes zoster. Using human cellular DNA microarrays, we found that many nuclear factor kappa B (NF-κB)-responsive genes were down-regulated in VZV-infected fibroblasts, suggesting that VZV infection inhibited the NF-κ B pathway. The activation of this pathway causes a cellular antiviral response, including the production of alpha/beta interferon, cytokines, and other proteins that restrict viral infection. In these experiments, we demonstrated that VZV interferes with NF-κB activation in cultured fibroblasts and in differentiated epidermal cells in skin xenografts of SCIDhu mice infected in vivo. VZV infection of fibroblasts caused a transient nuclear translocation of p50 and p65, the canonical NF-κB family members. In a process that was dependent upon the presence of infectious VZV, these proteins rapidly became sequestered in the cytoplasm of VZV-infected cells. Exclusion of NF-κB proteins from nuclei was associated with the continued presence of IκBα, which binds p50 and p65 and prevents their nuclear accumulation. IκBα levels did not diminish even though the protein became phosphorylated and ubiquitinated, as determined based on detection of the characteristic high-molecular-weight form of the protein, and the 26S proteasome remained functional in VZV-infected cells. VZV infection also inhibited the characteristic degradation of IκBα that is induced by exposure of fibroblasts to tumor necrosis factor alpha. As expected, herpes simplex virus 1 caused the persistent nuclear translocation of NF-κB proteins, which has been shown to facilitate its replication, whereas VZV infection progressed without persistent NF-κB nuclear localization. We suggest that VZV has evolved a mechanism to limit host cell antiviral defenses by sequestering NF-κB proteins in the cytoplasm, a strategy that appears to be unique among the herpesviruses. [ABSTRACT FROM AUTHOR]

The varicella-zoster virus (VZV) ORF62/63 intergenic region was cloned between the Renilla and firefly luciferase genes, which acted as reporters of ORF62 and ORF63 transcription, and recombinant viruses were generated that carried these reporter cassettes along with the intact native sequences in the repeat regions of the VZV genome. In order to investigate the potential contributions of cellular transregulatory proteins to ORF62 and ORF63 transcription, recombinant reporter viruses with mutations of consensus binding sites for six proteins within the intergenic region were also created. The reporter viruses were used to evaluate ORF62 and ORF63 transcription during VZV replication in cultured fibroblasts and in skin xenografts in SCIDhu mice in vivo. Mutations in putative binding sites for heat shock factor 1 (HSF-1), nuclear factor 1 (NF-1), and one of two cyclic AMP-responsive elements (CRE) reduced ORF62 reporter transcription in fibroblasts, while mutations in binding sites for HSF-1, NF-1, and octamer binding proteins (Oct-1) increased ORF62 reporter transcription in skin. Mutations in one CRE and the NF-1 site altered ORF63 transcription in fibroblasts, while mutation of the Oct-1 binding site increased ORF63 reporter transcription in skin. The effect of each of these mutations implies that the intact binding site sequence regulates native ORF62 and ORF63 transcription. Mutation of the only NF-κB/Rel binding site had no effect on ORF62 or ORF63 transcription in vitro or in vivo. The segment of the ORF62/63 intergenic region proximal to ORF63 was most important for ORF63 transcription, but mutagenesis also altered ORF62 transcription, indicating that this region functions as a bidirectional promoter. This first analysis of the ORF62/63 intergenic region in the context of VZV replication indicates that it is a dual promoter and that cellular transregulatory factors affect the transcription of these key VZV regulatory genes. [ABSTRACT FROM AUTHOR]