title: Selection and characterization of replicon variants dually resistant to thumb- and palm-binding nonnucleoside polymerase inhibitors of the hepatitis C virus.
abstract: 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.
authors.affiliation: Roche Palo Alto LLC, Palo Alto, CA 94304, USA.
authors.forename: Sophie
authors.initials: S
authors.lastname: Le Pogam
authors.forename: Hyunsoon
authors.initials: H
authors.lastname: Kang
authors.forename: Seth F
authors.initials: SF
authors.lastname: Harris
authors.forename: Vincent
authors.initials: V
authors.lastname: Leveque
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Giannetti
authors.forename: Samir
authors.initials: S
authors.lastname: Ali
authors.forename: Wen-Rong
authors.initials: WR
authors.lastname: Jiang
authors.forename: Sonal
authors.initials: S
authors.lastname: Rajyaguru
authors.forename: Gisele
authors.initials: G
authors.lastname: Tavares
authors.forename: Connie
authors.initials: C
authors.lastname: Oshiro
authors.forename: Than
authors.initials: T
authors.lastname: Hendricks
authors.forename: Klaus
authors.initials: K
authors.lastname: Klumpp
authors.forename: Julian
authors.initials: J
authors.lastname: Symons
authors.forename: Michelle F
authors.initials: MF
authors.lastname: Browner
authors.forename: Nick
authors.initials: N
authors.lastname: Cammack
authors.forename: Isabel
authors.initials: I
authors.lastname: Nájera
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002264:Carboxylic Acids; D024882:Drug Resistance, Viral; D004359:Drug Therapy, Combination; D014644:Genetic Variation; D016174:Hepacivirus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D020125:Mutation, Missense; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors*; D012093:Replicon / Q000235:genetics*; D012641:Selection, Genetic; D013876:Thiophenes / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors; D014779:Virus Replication
chemical_list: D000998:Antiviral Agents; D002264:Carboxylic Acids; D013876:Thiophenes; D017361:Viral Nonstructural Proteins; C550746:2-thiophene carboxylic acid; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase