Despite the eradication of smallpox, poxviruses continue to cause human disease around the world. At the core of poxvirus replication is the efficient and accurate synthesis and repair of the viral genome. The viral DNA polymerase is critical for these processes. Acyclic nucleoside phosphonate (ANP) compounds that target the viral polymerase are effective inhibitors of poxvirus replication and pathogenesis. Cidofovir (CDV) is an ANP that inhibits vaccinia virus (VAC) DNA polymerase (E9) DNA synthesis and 3-to-5 exonuclease (proofreading) activities. We determined that point mutations in the DNA polymerase genes of ANP-resistant (ANPR) VAC strains were responsible for CDV resistance and resistance to the related compound, HPMPDAP. Although these resistant strains replicated as well as wild-type VAC in culture, they were highly attenuated in mice. The generation of ANPR VAC strains, in combination with our knowledge of how CDV inhibits E9 activities, allowed us to study the hypothesized role of E9 in catalyzing double-strand break repair through homologous recombination. We provide evidence that VAC uses E9 proofreading activity to catalyze genetic recombination through single-strand annealing reactions (SSA) in infected cells. Both the polarity of end resection of recombinant intermediates and the involvement of polymerase proofreading activity establish these poxviral SSA reactions as unique among homologous recombination schemes. Furthermore, we identified roles for the VAC single-stranded DNA-binding (SSB) protein and nucleotide pools in regulating these reactions. During these later studies we uncovered a differential requirement for the large and small subunits of the VAC ribonucleotide reductase (RR) in viral replication and pathogenesis. Our studies suggest that poxviral RR small subunits form functional complexes with host large RR subunits to provide sufficient nucleotide pools to support DNA replication. We present a model whereby interaction of VAC SSB and RR proteins at replication forks allows for modulation of E9 activity through local nucleotide pool changes, which serves to maximize replication rates while still allowing for recombinational repair. / Virology
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:AEU.10048/860 |
| Date | 06 1900 |
| Creators | Gammon, Donald Brad |
| Contributors | Evans, David (Medical Microbiology and Immunology), Barry, Michele (Medical Microbiology and Immunology), Schang, Luis (Biochemistry), Reha-Krantz, Linda (Biological Sciences), Condit, Richard (Molecular Genetics and Microbiology, University of Florida) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 5901077 bytes, application/pdf |
| Relation | Gammon, D.B. and Evans, D.H. 2009. The 3-to-5 exonuclease activity of vaccinia virus DNA polymerase is essential and plays a role in promoting virus genetic recombination. Journal of Virology 83(9):4236-50., Gammon, D.B., Snoeck, R., Fiten, P., Krecmerova, M., Holy, A., De Clercq, E., Opdennaker, G., Evans, D.H., & Andrei, G. 2008. Mechanism of antiviral drug resistance of vaccinia virus: identification of residues in the viral DNA polymerase conferring differential resistance to anti-poxvirus drugs. Journal of Virology 82(24):12520-34., Hamilton, M.D., Nuara, A.A., Gammon, D.B., Buller, R.M., & Evans, D.H. 2007. Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase. Nucleic Acids Research 35(1):143-151., *Andrei, G., *Gammon, D.B., Fiten, P., De Clercq, E., Opdenakker, G., Snoeck, R., & Evans, D.H. 2006. Cidofovir resistance in vaccinia virus is linked to diminished virulence in mice. Journal of Virology 80(19):9391-9401. * Both authors contributed equally to this work |
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