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

Abstract

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/g 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.Hepatitis C virus (HCV) is an enveloped, positive-sense, single-stranded RNA virus of approximately 9.6 kb that possesses an RNA-dependent RNA polymerase (RdRp), NS5B. Like that in many RNA viruses, this RNA replicase lacks a proofreading mechanism. The mutation rate of the HCV RdRp is estimated to be 10

Preexisting drug-resistance mutations reveal unique barriers to resistance for distinct antivirals

“…After serial passaging in the presence of the NS5B polymerase inhibitor HCV-796, FACS selected a highly drug-resistant cell population. Sequencing this resistant cell line revealed the presence of the C316Y mutation, previously shown to confer resistance in vitro and identified in patients during treatment with HCV-796 (22,23). These results show that the fluorescent reporter gene is an accurate indicator of replicon levels and can be use to select drug-resistant mutants.…”

“…The S282T mutation is the only substitution reported to confer resistance to the nucleoside MK-608 and has significantly impaired fitness (11,29); accordingly, this compound showed the greatest barrier to resistance for any of the direct acting antivirals we tested. In contrast, multiple mutations can be selected by both A-782759 (H95Q, N411S, Y448H, and M414L/T) and HCV-796 (C316S/F/Y/N, S365A/T, L392F, and M414I/T/V), and these mutations range in fitness from significantly impaired to highly fit (17,18,23,29). Consistent with this knowledge, the resistance barrier curves for these nonnucleoside inhibitors demonstrate significant numbers of resistant foci persisting at high multiples of EC 50 .…”

Slow Binding Inhibition and Mechanism of Resistance of Non-nucleoside Polymerase Inhibitors of Hepatitis C Virus

“…Among the extensively investigated non-nucleosides documented to inhibit the RdRp activity of HCV NS5B, derivatives of various benzofuran and benzothiadiazine have been reported to bind to allosteric binding sites in the palm domain of NS5B (7,8). The palm domain, whose geometry is conserved in virtually all DNA and RNA polymerases, contains catalytic aspartic acids responsible for the nucleotidyl transfer reaction.…”

“…The structure of the NS5B apoenzyme and the NS5B-RNA complex reveals the characteristic right hand architecture of polymerase enzymes, comprising three distinct domains (palm, thumb, and finger) encircling the enzyme active site located in the palm domain (3-6). The structural and biochemical characterization of HCV NS5B polymerase can provide a basis for drug design efforts, and the elucidation of the mechanism of inhibition can guide the optimization of inhibitor efficiency against wild-type and resistant mutants.Among the extensively investigated non-nucleosides documented to inhibit the RdRp activity of HCV NS5B, derivatives of various benzofuran and benzothiadiazine have been reported to bind to allosteric binding sites in the palm domain of NS5B (7,8). The palm domain, whose geometry is conserved in virtually all DNA and RNA polymerases, contains catalytic aspartic acids responsible for the nucleotidyl transfer reaction.…”

“…Various in vitro studies using an HCV subgenomic replicon system have identified mutations that can confer resistance to inhibition by NNIs (2,8,16). Many of the mutations produce crossresistance to the same family of inhibitors, which will affect the design of optimal combination therapies.…”