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Journal of Virology, June 2006, p. 6146-6154, Vol. 80, No. 12
0022-538X/06/$08.00+0     doi:10.1128/JVI.02628-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Selection and Characterization of Replicon Variants Dually Resistant to Thumb- and Palm-Binding Nonnucleoside Polymerase Inhibitors of the Hepatitis C Virus

Sophie Le Pogam, Hyunsoon Kang, Seth F. Harris, Vincent Leveque, Anthony M. Giannetti, Samir Ali, Wen-Rong Jiang, Sonal Rajyaguru, Gisele Tavares,{dagger} Connie Oshiro, Than Hendricks, Klaus Klumpp, Julian Symons, Michelle F. Browner, Nick Cammack, and Isabel Nájera*

Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, California 94304

Received 16 December 2005/ Accepted 5 April 2006

Multiple nonnucleoside inhibitor binding sites have been identified within the hepatitis C virus (HCV) polymerase, including in the palm and thumb domains. After a single treatment with a thumb site inhibitor (thiophene-2-carboxylic acid NNI-1), resistant HCV replicon variants emerged that contained mutations at residues Leu419, Met423, and Ile482 in the polymerase thumb domain. Binding studies using wild-type (WT) and mutant enzymes and structure-based modeling showed that the mechanism of resistance is through the reduced binding of the inhibitor to the mutant enzymes. Combined treatment with a thumb- and a palm-binding polymerase inhibitor had a dramatic impact on the number of replicon colonies able to replicate in the presence of both inhibitors. A more exact characterization through molecular cloning showed that 97.7% of replicons contained amino acid substitutions that conferred resistance to either of the inhibitors. Of those, 65% contained simultaneously multiple amino acid substitutions that conferred resistance to both inhibitors. Double-mutant replicons Met414Leu and Met423Thr were predominantly selected, which showed reduced replication capacity compared to the WT replicon. These findings demonstrate the selection of replicon variants dually resistant to two NS5B polymerase inhibitors binding to different sites of the enzyme. Additionally, these findings provide initial insights into the in vitro mutational threshold of the HCV NS5B polymerase and the potential impact of viral fitness on the selection of multiple-resistant mutants.

* Corresponding author. Mailing address: Department of HCV Biology, Viral Diseases Therapeutic Area, Roche Palo Alto LLC, 3431 Hillview Ave., Palo Alto, CA 94304. Phone: (650) 855-5134. Fax: (650) 354-7554. E-mail: isabel.najera{at}roche.com.

{dagger} Present address: Novartis Institutes for Biomedical Research, Basel, Switzerland.

Journal of Virology, June 2006, p. 6146-6154, Vol. 80, No. 12
0022-538X/06/$08.00+0     doi:10.1128/JVI.02628-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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