Analysis of the function of two varicella-zoster virus proteins involved in gene regulation: IE63 and ORF29
Tanner, Tricia Lynn
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Varicella-zoster virus (VZV) is the causative agent of varicella during primary infection and zoster following reactivation from latency. During lytic infection, VZV controls expression of its genome in a temporally regulated fashion. Latent infection is characterized by expression of only a subset of viral genes, of which ORF63 and ORF29 are the most abundantly expressed. The IE63 and ORF29 proteins are important regulatory proteins in viral transcription; however, their mechanisms of action have not been fully elucidated. Further characterization of these proteins' mechanisms involved in viral and cellular gene expression is necessary for a complete understanding of the biology of VZV infection and was the aim of this work. It was found here via luciferase reporter assays that IE63 can upmodulate the activity of the important cellular EF-1[alpha] promoter in a cell type-dependent manner independent of other viral factors. This was the first report of a positive effect on a cellular promoter by IE63. This effect also correlated with increased EF-1[alpha] protein levels following viral infection, implying that VZV (via IE63) influences one or more of the many intracellular roles of EF-1[alpha]. Mutational studies showed that Region 2 of IE63 was important for its observed upmodulation of the activity of the viral transactivator, IE62, at the promoter of an important virulence factor, glycoprotein I (gI). Co-immunoprecipitation experiments also showed that IE63 associates with the ubiquitous cellular transcription factor, Sp1, though interaction did not appear to account for its observed upmodulatory effects in the context of model promoters. These data support the data in the current literature which suggest IE63 is a pleiotropic transcriptional modulator. The ORF29 protein, which primarily functions as the single-stranded DNA binding protein (SSB) during viral genome replication, was found to interact specifically and linearly with a cis-regulatory element within the gI promoter, designated the 29 Responsive Element (29RE), using electrophoretic mobility shift assays (EMSAs). T cell-specific proteins also bound to this viral sequence, and order of addition EMSAs showed that ORF29 altered the interaction of T cell-specific proteins with the 29RE. These data suggest a model in which ORF29 may potentially displace a cellular inhibitory factor which would then allow for its observed cell type-dependent upmodulation of IE62's transactivation of this viral promoter. In conclusion, this work supports the emerging concept that herpesvirus proteins play multifunctional roles in the myriad virus/host interactions that lead to infection, and that different cellular environments influence their activities.