title: Selection of clinically relevant protease inhibitor-resistant viruses using the genotype 2a hepatitis C virus infection system.
abstract: Treatment of patients infected with hepatitis C virus (HCV) with direct acting antivirals can lead to the emergence of drug-resistant variants that may pose a long-term threat to viral eradication. HCV replicons have been used to select resistance mutations; however, genotype 2a JFH-1-based viruses provide the opportunity to perform resistance selection in a bona fide infection system. In this study, we used a tissue culture-adapted J6/JFH-1 virus to select resistance to the NS3 protease inhibitors BILN-2061 and VX-950. Lunet-CD81 cells were infected with J6/JFH-1 virus and maintained in the presence of inhibitors until high-titer viral supernatant was produced. Viral supernatants were passaged over naive cells at escalating drug concentrations, and the resulting viruses were then characterized. Three NS3 resistance mutations were identified in BILN-2061-resistant viruses: A156G, D168A, and D168V. Interestingly, D168A, D168V, and A156T/V, but not A156G, were selected in parallel using a genotype 2a replicon. For VX-950, the T54A and A156S NS3 resistance mutations were identified in the virus selections, whereas only A156T/V emerged in genotype 2a replicon selections. Of note, VX-950 resistance mutations selected using the 2a virus (T54A and A156S) were also observed during VX-950 clinical studies in genotype 2 patients. We also performed viral fitness evaluations and determined that the mutations selected in the viral system did not confer marked reductions in virus production kinetics or peak titers. Overall, the HCV infection system is an efficient tool for drug resistance selections and has advantages for the rapid identification and characterization of clinically relevant resistance mutations.
authors.affiliation: Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404, USA. guofeng.cheng@gilead.com
authors.forename: Guofeng
authors.initials: G
authors.lastname: Cheng
authors.forename: Katie
authors.initials: K
authors.lastname: Chan
authors.forename: Huiling
authors.initials: H
authors.lastname: Yang
authors.forename: Amy
authors.initials: A
authors.lastname: Corsa
authors.forename: Maria
authors.initials: M
authors.lastname: Pokrovskii
authors.forename: Matthew
authors.initials: M
authors.lastname: Paulson
authors.forename: Gina
authors.initials: G
authors.lastname: Bahador
authors.forename: Weidong
authors.initials: W
authors.lastname: Zhong
authors.forename: William
authors.initials: W
authors.lastname: Delaney
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D024882:Drug Resistance, Viral / Q000235:genetics; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D011480:Protease Inhibitors / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000998:Antiviral Agents; D011480:Protease Inhibitors