Molecular mechanism of hepatitis C virus replicon variants with reduced susceptibility to a benzofuran inhibitor, HCV-796.
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 log(10) HCV copies/mug 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 A 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.
Antivral Discovery, Wyeth Research, 500 Arcola Road, Collegeville, Pennsylvania 19426, USA. ahowe@its.jnj.com
Anita Y M
AY
Howe
Huiming
H
Cheng
Stephen
S
Johann
Stanley
S
Mullen
Srinivas K
SK
Chunduru
Dorothy C
DC
Young
Joel
J
Bard
Rajiv
R
Chopra
Girija
G
Krishnamurthy
Tarek
T
Mansour
John
J
O'Connell
D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology*; D001572:Benzofurans / Q000037:antagonists & inhibitors*; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D024882:Drug Resistance, Viral; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D014644:Genetic Variation; D005838:Genotype; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors / Q000378:metabolism; D012093:Replicon / Q000187:drug effects* / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics
D000998:Antiviral Agents; D001572:Benzofurans; D004791:Enzyme Inhibitors; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C105430:benzofuran