Date of Award
Doctor of Philosophy (PhD)
Biochemistry and Molecular Biology
College of Graduate Studies
Poxviruses are large dsDNA viruses, unique in their cytoplasmic replication. This autonomy from the host nucleus poses novel challenges to genome replication, recombination and repair. Biochemical and genetic studies have confirmed that the viral genome encodes a repertoire of replication components (polymerase, processivity factor, single-strand DNA binding protein, primase/helicase, DNA ligase, scaffold protein). However, our knowledge of the mechanism of viral DNA replication remains incomplete, as is our understanding of which, if any, cellular proteins participate. We have shown that infection does not induce or dampen the cellular DNA damage response, but that a key component of this response, the ATR protein kinase, plays a role in viral genome replication. Inhibition of ATR destabilizes the small subunit (RRM2) of the cellular ribonucleotide reductase, delays the accumulation of nascent viral genomes, and results in the accumulation of subgenomic viral DNA fragments. To understand how vaccinia virus responds to exogenous DNA damage, we subjected cells to UV irradiation prior to (-1 hpi) or during (4 hpi) infection. Pre-irradiation of cells decreased protein synthesis and led to an ~12-fold reduction in viral yield. In addition to these cell-specific insults, irradiation at 4hpi introduced both CPD and 6-4-PP lesions into the viral genome, halted further DNA synthesis and led to a greater, ~35-fold reduction in viral yield. DNA lesions persisted throughout infection and were indeed present in the genomes encapsidated into nascent virions. Depletion of several components of the cellular nucleotide excision repair (XP-A, -F, -G) did not render infections hypersensitive to UV. However, infections performed with viruses lacking the cellular DNA ligase (A50) or structure specific nuclease (G5) were hypersensitive to UV irradiation (~3-fold and ~100-fold, respectively). When the DNA polymerase inhibitor araC was added to WT infections at the time of UV irradiation (4 hpi), hypersensitivity to UV irradiation was also seen (~10-fold). Virions produced under the latter condition contained elevated levels of CPD adducts, strongly suggesting that the viral polymerase can remove or repair UV lesions introduced into the viral genome.
Templeton, Conor W., "Vaccinia Virus DNA Metabolism: The Contributions of the Cellular Milieu and Novel Roles of the Viral DNA Replication Machinery Following Exogenous DNA Damage" (2022). MUSC Theses and Dissertations. 667.
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