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Antimicrobial Agents and Chemotherapy, September 2008, p. 3327-3338, Vol. 52, No. 9
0066-4804/08/$08.00+0     doi:10.1128/AAC.00238-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Molecular Mechanism of Hepatitis C Virus Replicon Variants with Reduced Susceptibility to a Benzofuran Inhibitor, HCV-796{triangledown}

Anita Y. M. Howe,1* Huiming Cheng,1 Stephen Johann,1 Stanley Mullen,1 Srinivas K. Chunduru,3,{dagger} Dorothy C. Young,3 Joel Bard,2 Rajiv Chopra,2 Girija Krishnamurthy,2 Tarek Mansour,2 and John O'Connell1

Antivral Discovery, Wyeth Research, 500 Arcola Road, Collegeville, Pennsylvania 19426,1 Chemical Screening Sciences, Wyeth Research, Cambridge, Massachusetts 02140,2 ViroPharma Incorporated, 405 Eagleview Boulevard, Exton, Pennsylvania 193413

Received 20 February 2008/ Returned for modification 2 April 2008/ Accepted 4 June 2008

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 to 4 log10 HCV copies/µg total RNA (the concentration of the compound that inhibited 50% of the HCV RNA level was 9 nM). 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 impacts of the amino acid substitutions on viral fitness and drug susceptibility were examined in recombinant replicons and NS5B enzymes with the single-amino-acid mutations. The replicon variants were 10- to 1,000-fold less efficient in forming colonies in cells than the wild-type replicon; the S365L variant failed to establish a stable cell line. Other variants (L314F, I363V, and M414V) had four- to ninefold-lower steady-state HCV RNA levels. Reduced binding affinity with HCV-796 was demonstrated in an enzyme harboring the C316Y mutation. The effects of these resistance mutations were 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 susceptibilities to pegylated interferon, ribavirin, and other HCV-specific inhibitors. The combined virological, biochemical, biophysical, and structural approaches revealed the mechanism of resistance in the variants selected by the potent polymerase inhibitor HCV-796.

* Corresponding author. Present address: Tibotec, Gen. De Wittelaan L11 B3 2800 Mechelen, Belgium. Phone: 32 15 46 1035. Fax: 32 15 46 1936. E-mail: ahowe{at}its.jnj.com

{triangledown} Published ahead of print on 16 June 2008.

{dagger} Present address: TetraLogic Pharmaceuticals, 365 Phoenixville Pike, Malvern, PA 19355.

Antimicrobial Agents and Chemotherapy, September 2008, p. 3327-3338, Vol. 52, No. 9
0066-4804/08/$08.00+0     doi:10.1128/AAC.00238-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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