Figure 7

Impact of various substitutions on NS5A L31 and Y93 in HCV replication and responsiveness to DCV and HA1077. (a) DCV resistance-associated substitutions in NS5A of HCV GT1a/b and GT2a strains. Shown in black and blue are the amino acid substitutions most frequently identified in the past, and in red the previously uncovered residues identified in this study. The residues shown in parenthesis are the mutations identified previously in GT2a but not in this study with JFH1. (b) Susceptibility of JFH1 derivatives with the indicated substitutions to HA1077 and DCV. Antiviral activity of HA1077 (20 μM) and DCV (100 pM) against wild-type JFH1 and its various derivatives was assessed by quantification of HCV RNA titer by RT-qPCR 3 days after transfection of viral RNA transcripts into Huh7 cells. Data are representative of three independent experiments, each involving triplicates. The significance of differences in means between groups was analyzed by one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test. ***P ≤ 0.001; n.s, not significant. (c) Replication competence of the transfected HCV transcripts described in (b) was assessed by northern blotting. Shown at the top is a representative blot. HCV blots were quantified using a PhosphorImager and normalized to GAPDH expression. Shown at the bottom panel are the relative HCV replication levels (mean ± s.d.) estimated from two independent experiments. **P ≤ 0.01 by unpaired Student’s t-test. (d,e) Infectivity of JFH1 derivatives bearing a DCV resistance-associated NS5A substitution. Huh7 cells were infected with an equal volume of culture supernatants from HCV RNA-transfected cells recovered at 3 days post-transfection, prior to immunostaining of HCV core protein (Core; green) 2 days later. DAPI, nuclear staining (blue) (d). Relative percentages of Core-positive cells in HCV-infected Huh7 cells (e). The data shown are the means ± s.d. (relative infectivity of each mutant), obtained from three technical replicates. Scale bar, 50 μm.