Protein-ligand and protein-protein interactions involved in de novo initiation of RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp)

Hepatitis C Virus (HCV) is a major etiological agent of non-A, non-B chronic hepatitis. An estimated 180 million people worldwide are infected with this virus. Given this huge epidemic and relative low efficacy of current treatment for hepatitis C, it is urgent to develop better antiviral therapeutics. Better understanding of the mechanism of all aspect of RNA-dependent RNA synthesis will contributes for this purpose. A feature unique to RNA viruses is the initiation of RNA synthesis from the 3' terminus of the template by a de novo mechanism. This dissertation research is focused on the characterization of the interactions between the HCV RdRp and ligands (or proteins) that are involved during de novo initiation of RNA synthesis by the HCV RdRp.; Fluorescence spectroscopy was used to examine the equilibrium binding of ligands by the HCV RdRp. Three independent intrinsic and extrinsic fluorophores were used to characterize protein-ligand and protein-protein interactions. Using these fluorescence assays, we determined that the HCV RdRp had an approximately 9-fold higher affinity for Mn2+ than for Mg2+. Furthermore, the minimal length of RNA required for stable binding to the HCV RdRp was determined to be seven nucleotides. HCV RdRp bound to de novo initiation competent RNA with ∼2 fold enhanced affinity in the presence of initiation specific GTP and Mn2+ ion, suggesting a modest stabilization effect exists during the formation of a productive initiation ternary complex.; Mapping of the RNA contact sites on the HCV RdRp were performed by using reversible formaldehyde cross-linking coupled with RNA affinity chromatography, and mass spectrometric analyses. Identified cross-linked peptides are located in the fingers subdomain, connecting loops of fingers and thumb subdomains and active site cavity. The identified regions contained all predicted RNA contacting residues from the crystal structures. Mutational analysis of the HCV RdRp at several likely residues presumed to be important in contacting RNA decreased RNA synthesis in vitro. When the HCV replicons containing the mutations were tested in cultured liver cells, all but one mutation prevented the replication, indicating that the sites mapped are important for HCV RNA replication.; The interaction between the HCV RdRp with itself and with the HCV NS3 protein was also examined using biochemical and biophysical approaches. Altogether, this work contributes to understanding of mechanism of RNA synthesis by the HCV RdRp.