| Poxviruses replicate entirely in the cytoplasm of eukaryotic cells. This unusual compartmentalization obligates poxviruses to encode the machinery necessary for the independent replication and expression of their genome. This thesis examines two distinct facets of poxvirus replication.; The 192-kb linear DNA genome of vaccinia, the prototypic poxvirus, has covalently closed hairpin termini that contain twelve extrahelical bases. Vaccinia telomeres have previously been implicated in the initiation of viral genome replication. To further understand the role of telomeres during viral infection, electrophoretic mobility shift assays were used to examine specific protein complexes that form on telomeric DNA. Telomere-binding activity was observed using extracts prepared from virions and from the cytoplasm of infected cells. Significantly, viral hairpins lacking extrahelical bases did not form DNA/protein complexes. Competition experiments have shown that complex formation requires at least two extrahelical bases but not the hairpin turnaround.; Using affinity purification, the vaccinia I1 and I6 proteins were identified as being responsible for telomere-binding activity. A virally encoded protein that nicks viral hairpins was also identified, and found to be the vaccinia K4 protein. These three viral proteins are likely to contribute to some aspect of genome replication and/or encapsidation.; Another focus of this thesis was an investigation of the vaccinia H5 phosphoprotein. H5 is the only known viral substrate of the vaccinia B1 protein kinase, which has been implicated in playing a role in viral DNA replication. To further understand the role of B1, a genetic analysis of its substrate, H5, was performed. A temperature-sensitive virus with a lesion in the H5 gene (tsH5) was engineered using “charged-cluster-to-alanine” mutagenesis. Surprisingly, no defect in viral DNA replication or gene expression was observed in tsH5 infections. By electron microscopy, a profound defect in virion morphogenesis was observed in tsH5 infections under non-permissive conditions. Specifically, there were no signs of normal virion particles or precursors, and viral factories appeared abnormal. Interestingly, protein produced from the mutant H5 allele is stable at the non-permissive temperature and exerts a dominant phenotype. These data implicate H5 in playing an important and novel role in an early stage of viral morphogenesis. |
