Abstract
The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) is a key target for antiviral intervention. The goal of this study was to identify the binding site and unravel the molecular mechanism by which natural flavonoids efficiently inhibit HCV RdRp. Screening identified the flavonol quercetagetin as the most potent inhibitor of HCV RdRp activity. Quercetagetin was found to inhibit RdRp through inhibition of RNA binding to the viral polymerase, a yet unknown antiviral mechanism. X-ray crystallographic structure analysis of the RdRp-quercetagetin complex identified quercetagetin's binding site at the entrance of the RNA template tunnel, confirming its original mode of action. This antiviral mechanism was associated with a high barrier to resistance in both site-directed mutagenesis and long-term selection experiments. In conclusion, we identified a new mechanism for non-nucleoside inhibition of HCV RdRp through inhibition of RNA binding to the enzyme, a mechanism associated with broad genotypic activity and a high barrier to resistance. Our results open the way to new antiviral approaches for HCV and other viruses that use an RdRp based on RNA binding inhibition, that could prove to be useful in human, animal or plant viral infections.
Highlights
Hepatitis C virus (HCV) is a member of the Hepacivirus genus within the Flaviviridae family
We previously demonstrated that silibinin’s antiviral effect is at least partly explained by its ability to act as a direct non-nucleoside inhibitor of HCV RNA-dependent RNA polymerase (RdRp) activity [20]
Our in vitro enzyme assay based on RNA duplex formation in the presence of HCV-nonstructural 5B (NS5B) 21 allowed us to identify the flavonols, and among them quercetagetin, as the most potent inhibitors of HCV RdRp among the flavonoids tested
Summary
Hepatitis C virus (HCV) is a member of the Hepacivirus genus within the Flaviviridae family. Chronic HCV infection is responsible for chronic hepatitis which, in turn, leads to cirrhosis in ∼20% of cases and hepatocellular carcinoma at an incidence of 4–5% per year in cirrhotic patients [1]. IFN-free regimens yielding high HCV infection cure rates (over 90%) are likely to reach the market in 2014– 2015 and onwards. These new treatment regimens will, be extremely costly and will generate multidrug resistance in patients who fail on therapy. They are unlikely to be available in the short- to mid-term in many areas of the world where therapeutic needs are high
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