Figure 1

Identification of a small molecule that inhibits HCV replication.

(a) The chemical structure of RO8191. (b) After treatment with various concentrations of RO8191 or 100 IU/mL IFN-α for 72 h, HCV replication levels were examined using a luciferase assay (left graph) and cell viabilities were determined using a WST-8 assay (right graph). The mean values and their SDs were recorded for treated cells as a percentage of the values for untreated cells and the values represent the means of 3 independent experiments. (c) Total RNA was extracted from HCV replicon cells cultured with the indicated concentration of RO8191 or 100 IU/mL IFN-α for 72 h; HCV RNA levels were analyzed using real-time RT-PCR. The mean values and their SDs were recorded for treated cells relative to the mRNA levels of β-actin and are shown as a percentage of untreated cells. The values represent the means of 3 independent experiments. (d) HCV replicon cells were treated with control medium (left panels) or 10 μM RO8191 (right panels) for 24 h and immunostained with Hoechst 33452 (blue), anti-NS3 antibody (green) and anti-NS4A antibody (red). The results were then merged (yellow). (e) HCV replicon cells were treated with the indicated concentrations of RO8191 or 100 IU/mL IFN-α for 72 h. Whole cell lysates were immunoblotted with antibodies specific to the indicated HCV NS proteins. (f) After infection with the HCV JFH1 strain, Huh-7/K4 cells were treated with the indicated concentrations of RO8191 for 72 h. Total RNA was extracted and the HCV RNA levels were analyzed using quantitative real-time RT-PCR.