The RNA-dependent RNA polymerase (RdRp) of viruses belonging to the Flaviviridae family, including the hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) is critical for viral replication. The major goal of this PhD study was to biochemically characterize the role of the polymerase during initiation and elongation of RNA synthesis, utilizing the BVDV RdRp as a model system. We showed that the BVDV polymerase efficiently incorporates chain-terminating nucleoside analogues, which ultimately arrest RNA synthesis. The incorporated chain-terminators, however, can be removed from the primer terminus in the presence of pyrophosphate (PPi). These results suggest that the phosphorolytic excision of incorporated chain-terminators is a possible mechanism that can diminish the efficiency of this class of compounds against viral RdRps. The chain-terminators then served as valuable tools in subsequent experiments to analyze the functional role(s) of the RdRp-associated GTP-specific binding site (G-site) and the consequences of GTP binding during the initiation of RNA synthesis. The results provide biochemical evidence for the existence of a G-site in the BVDV enzyme, and suggest that GTP binding controls template positioning during de novo initiation. Finally, through the development of a novel ribonuclease-based footprinting assay, it was determined that catalytically active complexes contact the newly synthesized RNA during elongation of RNA synthesis with approximately 6-7 base pairs. The polymerase moves along the template according to the position where RNA synthesis is arrested. Taken together, this study provides novel insight into mechanisms involved during initiation and elongation of RNA replication of viruses belonging to the Flaviviridae family. The ability of RdRps to excise incorporated chain-terminators points to possible shortcomings of nucleoside analogue inhibitors that are under development as antiviral agents for the treatment of infection with HCV.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.111870 |
| Date | January 2006 |
| Creators | D'Abramo, Claudia M. |
| Publisher | McGill University |
| 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 | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002584920, proquestno: AAINR32346, Theses scanned by UMI/ProQuest. |
Page generated in 0.0019 seconds