FIG 3.

Piperazinylbenzenesulfonamides inhibit a late step of HCV entry for all the major HCV genotypes. (A) Huh-7 cells were treated for 3 h with SB258585 at different concentrations, concomitant with HCVpp JFH1 or RD114pp incubation. At 48 h postinfection, cells were lysed and luciferase activity measured in order to quantify the infection. (B) Huh-7 cells were incubated for 2 h with SB258585 at different concentrations, along with a GFP-expressing adenovirus. The percentage of infection was determined by quantifying GFP-positive cells at 24 h postinfection. (C) Primary human hepatocytes (PHHs) were treated for 2 h with SB258585 at different concentrations, in addition to infection with HCVcc JFH1. Infection was quantified by qRT-PCR at 24 h postinfection. (D) Huh-7.5 cells were treated for 3 h with SB258585, in addition to infection with JFH1-based HCV genotype 1 to 6 recombinant viruses with strain-specific core-NS2. Infection was quantified by immunofluorescence assay at 48 h postinoculation. Error bars represent standard deviations (SD). (E) After 1 h of viral attachment at 4°C, Huh-7 cells were shifted to 37°C and treated with SB258585 at the indicated concentrations, following 15-min kinetics for 2 h. Proteinase K (50 μg/ml) and bafilomycin A (25 nM) were used as controls for early and late entry steps, respectively. Infection was quantified at 30 h postinfection by immunofluorescence assay, and the value for each time point was normalized to that for the corresponding DMSO condition. (F) Chemical structures of SB258585 and SB399885. (G) Huh-7 cells were treated with SB399885 at different concentrations for 2 h prior to, during, or after HCVcc JFH1 incubation, following the schematized kinetics. Infection was quantified at 30 h postinfection by immunofluorescence assay. (H) Huh-7 cells were treated with SB399885 for 2 h, followed by 28 h of rest. An MTS assay was performed in order to evaluate cell toxicity. (I) After 1 h of viral attachment at 4°C, Huh-7 cells were shifted to 37°C and treated with SB399885 at the indicated concentrations, following 15-min kinetics for 2 h. Proteinase K (50 μg/ml) and bafilomycin A (25 nM) were used as controls for early and late entry steps, respectively. Infection was quantified at 30 h postinfection by immunofluorescence assay, and the value for each time point was normalized to that for the corresponding DMSO condition. Results are presented as means ± SEM (n = 3 [A to D, G, and H] and n = 2 [E and I]). PHH results are presented as means for triplicates ± SEM for two independent experiments. One-way (B, C, and H) or two-way (A and G) ANOVA followed by the Dunnett or Bonferroni posttest was performed for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.001; ns, nonsignificant.