Hepatitis C virus (HCV) infects 2.7 million Americans and is a leading cause of chronic hepatitis and hepatocellular carcinoma. It is treated with interferon ? plus the nucleoside analog ribavirin. A controversial mechanism for ribavirin is through incorporation into the viral genome by the HCV RNA polymerase (RdRp) following phosphorylation to ribavirin triphosphate by cellular enzymes. Incorporation of ribavirin into HCV RNAs would reduce viral fitness and inhibit subsequent rounds of RNA synthesis. In a small pilot study we found that natural sequence variation in the HCV RdRp led to increased use of guanosine relative to uracil (G/U ratio) during RNA synthesis in 2 of 4 responders to therapy but in none of the 4 non-responders due to elevated GTP use rather than decreased UTP use. This observation has direct clinical implications because ribavirin is a guanosine analog. Hypothesis: Sequence variation in the HCV RdRp leads to variable guanosine and/or ribavirin use during RNA synthesis and modulates success of therapy employing ribavirin.
Aim 1. Determine if high G/U ratios are associated with success of antiviral therapy. The RdRps with high G/U ratios were from patients who failed interferon ? monotherapy and were then retreated with interferon ? plus ribavirin. This selected for patients whose response to treatment was primarily due to addition of ribavirin, but it precluded associating high guanosine use with response to therapy in treatment- naive patients. Therefore, we will measure the G/U ratios of RdRps from participants in the Virahep-C trial of therapy for HCV to determine if G/U ratios correlate with success of therapy in treatment-naive patients.
Aim 2. Determine the relationship of high G/U ratios and use of ribavirin triphosphate. RdRps with high G/U ratios are predicted to incorporate ribavirin into HCV RNAs at higher rates than RdRps with low G/U ratios. Therefore, we will measure in vitro use of ribavirin triphosphate as a substrate by recombinant RdRps from Aim 1 and correlate this activity with response to therapy.
Aim 3. Assess effects of variations associated with altered guanosine and/or ribavirin use on the RdRp structure. Molecular modeling will be used to identify RdRp variations that may cause altered nucleotide use. Key variations predicted to elevate guanosine or ribavirin use will be transferred to RdRps with low G/U ratios and guanosine and ribavirin use will be measured to test the structural predictions. These studies will characterize how natural variation in the HCV RdRp affects its ability to use guanosine and ribavirin during RNA synthesis and will determine if elevated guanosine or ribavirin use correlates with success of HCV therapy. Associating success of anti-HCV therapy with use of ribavirin by the RdRp would provide strong evidence for ribavirin's mechanism in humans as being through incorporation into the viral genome and would resolve the controversy of how ribavirin contributes to HCV clearance. ? ? ?
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