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Antimicrob. Agents Chemother. doi:10.1128/AAC.00238-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

MOLECULAR MECHANISM OF HEAPTITIS C VIRUS REPLICON VARIANTS WITH REDUCED SUSCEPTIBILITY TO A BENZOFURAN INHIBITOR, HCV-796

Antivral Discovery, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426; Chemical Screening Sciences, Wyeth Research, Cambridge, MA, 02140; ViroPharma Incorporated, 405 Eagleview Boulevard, Exton, PA 19341

^* To whom correspondence should be addressed. Email: howeaym{at}wyeth.com.

	Abstract

HCV-796 selectively inhibits hepatitis C virus (HCV) NS5B RNA dependent RNA polymerase. In hepatoma cells containing a genotype 1b HCV replicon, HCV-796 reduced HCV RNA levels by 3-4 log₁₀ HCV copies/µg total RNA (EC₅₀ = 9nM). Cells bearing replicon variants with reduced susceptibility to HCV-796 were generated in the presence of HCV-796 followed by G418 selection. Sequence analysis of the NS5B gene derived from the replicon variants revealed several amino acid changes within 5 Å of the drug-binding pocket. Specifically, mutations were observed at Leu314, Cys316, Ile363, Ser365 and Met414 of NS5B, which directly interact with HCV-796. The impact of the amino acid substitutions on viral fitness and drug susceptibility was examined in recombinant replicons and NS5B enzymes with the single amino acid mutations. The replicon variants were 10- to 1000-fold less efficient in forming colonies in cells compared with the wild-type replicon; the S365L variant failed to establish a stable cell line. Other variants (L314F, I363V and M414V) had 4- to 9-fold lower steady state HCV RNA levels. Reduced binding affinity with HCV-796 was demonstrated in an enzyme harboring the C316Y mutation. The effect of these resistance mutations was structurally rationalized using x-ray crystallography data. While different levels of resistance to HCV-796 were observed in the replicon and enzyme variants, these variants retained their susceptibility to pegylated interferon, ribavirin, and other HCV specific inhibitors. The combined virological, biochemical, biophysical and structural approaches have revealed the mechanism of resistance in the variants selected by the potent polymerase inhibitor, HCV-796.