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/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.

FIG. 1.

Structure of HCV-796, 5-cyclopropyl-2-(4-fluorophenyl)-6-[(2-hydroxyethyl)(methanesulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide.

FIG. 2.

Multiple treatments of clone A cells with HCV-796. Clone A cells were treated with 0.1 μM and 1 μM of HCV-796 in Dulbecco's minimal essential medium containing 2% fetal calf serum, 0.5% DMSO, and no G418. Control cells were grown in the same medium without HCV-796. When the cell density reached about 80% confluence, the cells were split, and aliquots of cells were harvested for total cellular RNA extraction. The amounts of HCV RNA and rRNA were determined in a quantitative duplex TaqMan RT-PCR assay. The y axis represents HCV copies per μg of total cellular RNA (using rRNA as a marker for quantification). Each data point represents an average of three cell replicates. The error bars indicate standard deviations.

FIG. 3.

Effect of HCV-796 on variant cells selected by HCV-796. Seven thousand clone A or 796R cells were seeded per well in a 96-well tissue culture dish and treated with increasing concentrations of HCV-796 in the absence of G418. Cells were harvested 3 days after treatment and analyzed for HCV and rRNAs using a quantitative duplex TaqMan RT-PCR. The numbers of HCV RNA copies per μg total RNA were compared with those in the control cells. The data shown in the graph are the results from 1 of the 12 independent experiments. Each point represents an average of four replicates. The EC₅₀ in the replicon-containing cells is indicated. The error bars indicate standard deviations.

FIG. 4.

Crystal structure showing HCV-796-associated amino acid mutations. (A) Overview of the HCV NS5b protein bound to HCV-796. The compound is shown with yellow carbons. The thumb domain is shown in blue, the palm in gold, and the fingers in green. The serine-rich loop is shown in orange (just in front of the compound), the active-site loop is shown in cyan, helix G is shown in salmon, and helix M is shown in dark blue. (B) Details of the HCV-796 binding site. The image is rotated 90° clockwise from panel A. HCV-796 is shown in yellow in the center of the model. The serine-rich loop is shown in orange, the active-site loop is shown in cyan, and helix G is shown in salmon. The hydrogen bond between the amide nitrogen of HCV-796 and the OH of Ser 365 is shown in blue. The positions of key wild-type amino acids are highlighted.

FIG. 5.

(A) Amino acid changes in NS5B derived from clone A control cells. (B) Amino acid changes in NS5B derived from 796R(0.1 μM) and 796R(1 μM) cells. (C) Amino acid changes in 796R(10 μM) cells. Some amino acids in the replicons are colored to illustrate the linkage of mutations. Replicon variants with only one mutation within NS5B are shaded in grey.

FIG. 5.

(A) Amino acid changes in NS5B derived from clone A control cells. (B) Amino acid changes in NS5B derived from 796R(0.1 μM) and 796R(1 μM) cells. (C) Amino acid changes in 796R(10 μM) cells. Some amino acids in the replicons are colored to illustrate the linkage of mutations. Replicon variants with only one mutation within NS5B are shaded in grey.

FIG. 6.

Binding of HCV-796 with NS5B. Shown is the binding isotherm of the interaction of HCV-796 with the wild-type NS5B and mutant NS5B bearing the C316Y substitution (NS5B C316Y). HCV-796 was incubated with the enzyme according to the procedure described in Materials and Methods. The change in the intrinsic fluorescence of HCV-796 at 375 nm, F0-F, in the presence of increasing concentrations of either the wild-type or mutant NS5B enzyme is proportional to the bound inhibitor concentration. The solid line corresponds to the curve fit to the quadratic equation.