Role of the varicella zoster virus ORF9 protein in viral replication
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The varicella zoster virus (VZV) ORF9 protein is a member of the herpesvirus UL49 gene family but shares limited identity and similarity with the UL49 prototype, HSV-1 VP22. ORF9 mRNA is the most abundantly expressed message during VZV infection, however little is known concerning the functions of ORF9 protein. In the work performed in this thesis, it was found that the VZV major transactivator, IE62, and the ORF9 protein can be co-precipitated from infected cells. Yeast two hybrid analysis localized the region of the ORF9 protein required for interaction with IE62 to amino acids 117-186. Protein pull down assays showed that amino acids 1-43 of the acidic transcriptional activation domain of IE62 bind ORF9 protein. Confocal microscopy showed that in the absence of other viral proteins ORF9 protein localizes in the cytoplasm while IE62 localizes in the nucleus. In VZV-infected cells, the ORF9 protein localized to the cytoplasm whereas IE62 exhibited both nuclear and cytoplasmic localization. Co-immunoprecipitation and confocal microscopy indicate the presence of ORF9-IE62-tubulin complexes in infected cells. RNA interference experiments showed that ORF9 is important for cell-to-cell spread of VZV and/or syncytium formation during infection. These data suggest a model for ORF9 protein function, involving complex formation with IE62 and possibly other tegument proteins in the cytoplasm at late times in infection allowing efficient delivery of viral proteins upon fusion of infected with uninfected cells as well as incorporation of IE62 into VZV virions. Factors involved in expression of the ORF9 gene were also examined. Analysis of the ORF8/ORF9 intergenic region by truncation and site-specific mutagenesis indicates that the minimal ORF9 promoter is made up of a consensus binding site for the cellular factor USF and a TATA element. Using a bidirectional reporter vector, it was found that the relative promoter strength in the ORF9 direction was maximally four times greater than that in the ORF8 direction. The possibility that the ORF9 protein stabilizes its own message was also investigated. Nitrocellulose filter binding experiments showed that the ORF9 protein associates with RNA. However, no significant difference in binding was observed using RNA oligomers derived from several VZV genes. Co-immunoprecipitation of infected cell RNAs and ORF9 protein also showed no specificity for ORF9 mRNA. Thus, the high levels of expression reported for the ORF9 gene relative to ORF8 and other VZV genes is in part due to relative promoter strength but also involves as yet unidentified mechanisms.