FIG 4.

Sensitivities of NS3 variants to simeprevir. (A) wt HCV RNA or the HCV RNAs encoding NS3 variants, such as Q80K, Q80R, R155K, D168A, D168V, Q80K+R155K, or Q80R+R155K in H77S.3/GLuc2A, were transfected into Huh-7.5 cells. The medium was replaced with fresh medium containing only DMSO or simeprevir at 100, 200, 300, 400, 500, 750, and 1,000 nM after 24 h and at 24-h intervals thereafter. The secreted GLuc activity in the medium was determined at 72 h posttransfection. In each NS3 variant, the GLuc activity in simeprevir-treated cells was normalized to that in cells treated with DMSO only. Statistical significance was analyzed in comparison to 0 nM simeprevir by one-way ANOVA and Bonferroni's multiple-comparison tests. (B) HCV RNAs encoding the Q80K+R155K and Q80R+R155K variants in H77S.3/GLuc2A were transfected into Huh-7.5 cells. The medium was replaced with fresh medium containing only DMSO or simeprevir at 100, 500, 750, 1,000, 2,000, 3,000, 4,000, 5,000, and 10,000 nM at 24 h and at 24-h intervals thereafter. The secreted GLuc activity was determined at 72 h posttransfection. For each NS3 variant, the GLuc activity from simeprevir-treated cells was normalized to that of cells treated with DMSO only. Statistical significance was analyzed in comparison to 0 nM simeprevir by one-way ANOVA and Bonferroni's multiple-comparison tests. (C) HCV RNAs encoding the Q80K+R155K or Q80R+R155K variant in H77S.3 were transfected into Huh-7.5 cells. The medium was replaced with fresh medium containing only DMSO or simeprevir at 100, 250, 500, 750, 1,000, and 5,000 nM after 24 h. Total cell lysates were collected 96 h after transfection and then probed by Western blotting with anticore or β-actin antibody. *, P < 0.05; **, P < 0.01; ***, P < 0.001.