Graduate Thesis Or Dissertation
 

The regulation of a vaccinia virus late gene required for morphogenesis

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  • Vaccinia virus gene expression is characterized by tight temporal regulation. Viral early transcription begins within the cytoplasm upon infection; three hours later, early genes are repressed, DNA replication occurs and late gene expression commences. As an approach toward understanding the mechanisms governing vaccinia late gene expression, I have undertaken an in vivo analysis of six vaccinia late promoters. The promoters were identified by abutting putative vaccinia promoter sequences to a reporter gene and assaying for reporter gene activity during transient expression. Once biologically active promoter sequences were identified, the L65 and ORF Al promoters were subjected to 5' deletion mutagenesis to define active regions. At this point, attention focused on the L65 promoter due to the added complexity of two differentially regulated RNA start sites. Additional 5' deletion mutagenesis pinpointed regions responsible for transcription from these sites. The availability of these cloned small promoter fragments facilitated assays for vaccinia late promoter specific binding factors. The discovery of a protein factor which binds specifically to the L65 promoter suggests that this DNA binding protein may also be a transcription factor. This promoter drives the expression of a major late polypeptide of unknown function (L65); to determine if post-transcriptional regulatory mechanisms affect L65 expression and to localize L65 during the infection cycle, L65 specific anti-sera was prepared against a trpE/L65 fusion protein expressed in E. coli. This reagent, together with the information obtained regarding the cis-acting elements and trans-acting factors involved in the transcriptional regulation of the L65 promoter will allow a directed approach toward understanding the regulation of L65 gene expression and perhaps the function of L65.
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