Analysis of the regulation of a bi-directional varicella-zoster virus (VZV) promoter: The ORF 28/29 promoter
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Varicella-Zoster Virus (VZV) is a member of the alphaherpesviridae and the causative agent of two human diseases: chicken pox (varicella) and shingles (zoster). Primary systemic infection is followed by latent infection in the dorsal root ganglia. All 70 open reading frames (ORF) encoded by the VZV genome are believed to be expressed during productive infection. However, only a small number of VZV genes are expressed in latency. Regulation of VZV gene expression at the transcriptional level is the topic of this thesis. Work has focused on an intergenic divergent regulatory element, the ORF28/29 promoter, and its activation by the major viral transactivator, IE62. This region was chosen based on the observation that both genes are expressed during VZV lytic infection, but only the ORF 29 gene is expressed in latently infected neurons. In the first part of this thesis work, promoter elements within the ORF 28/29 intergenic region were delinaeated in the context of IE62 activation in a melanoma cell line, and a neuroblastoma cell line. Analysis of the functional elements within the regulatory region revealed that a fusion of two unidirectional promoters which share an essential USF binding site but contain distinct TATA elements. A single TATA element directs expression in the ORF 28 direction whereas two TATA elements direct ORF 29 gene expression and are alternatively and differentially utilized for transcription initiation. An Sp1 site localized proximal to the ORF 28 gene which functions as an activator element for expression in both directions was also identified. Similar results were derived in both cell types in terms of the polarity and the architecture of the promoter in the context of IE62 activation. These results indicate that the ORF 28 and ORF 29 genes can be expressed either coordinately or independently and that the observed expression of only the ORF 29 gene during VZV latency may involve neuron specific cellular factors and/or structural aspects of the latent viral genome. The functional interplay between USF and IE62, the two essential trans-acting factors in activation of the ORF28/29 regulatory element was analyzed in the second part of this thesis work. This work showed that the activation domain of USF is both necessary and sufficient to mediate IE62 activation. However, the direct physical interaction between IE62 and USF which was mapped to as 238-258 of IE62 and the bHLH-zip domain of USF is dispensable for their synergistic promoter activation. IE62 binding to the promoter is independent of USF binding but is involved in stabilizing TBP binding. These results indicate that USF mediated IE62 activation is a result of synergistic promoter activation through two transcriptional activation domains that may independently interact with the elements of the general cellular transcription apparatus. This work advanced understanding of the regulation of the VZV ORF28/29 regulatory element which allowed insight into possible mechanisms of differential VZV gene expression during productive and latent infection. The functional interaction between USF and IE62 exhibited a novel form of synergy between a cellular transcription factor and a viral transactivator independent of a direct physical interaction.