FIG 9.

YM-53601 inhibits HCV RNA replication of HCV JFH-1. (A) Transient-replication assay using JFH-1 subgenomic replicons. Huh-7.5.1-8 cells were transfected with SGR-JFH1/Luc (closed symbols) or SGR-JFH1/Luc-GND (open symbols) RNAs by electroporation and then placed in serum-free medium. At 5 h posttransfection, YM-53601 (final concentration, 1.5 μM) (squares and dashed line) or DMSO (circles and solid line) was added to the medium. The cells were harvested at the indicated time points (posttransfection) and assayed for luciferase activity. (B and C) Huh-7.5.1-8 cells were pretreated with 1.5 μM YM-53601 or DMSO in serum-free medium for 42 h and then transfected with SGR-JFH1/Luc-GND RNA by lipofection. After transfection, the cells were further treated in the same medium and harvested at the indicated time points. (B) The cells were lysed and assayed for luciferase activity. (C) An equal amount of protein (10 μg/lane) in each cell lysate was subjected to immunoblotting for NS3 and GAPDH proteins, and each protein band was quantified. The relative amount of NS3 protein was calculated by dividing its intensity by that of GAPDH protein in the same lane. Data are expressed as a percentage of the relative amount at 9 h posttransfection. The value at 9 h posttransfection was not significantly different between the drug-treated and untreated cells (data not shown). Data in each graph are means ± SD for triplicate samples from one representative experiment. Similar results were obtained in at least two independent experiments. Statistical analysis was performed between drug-treated and control cells harboring the same replicon. *, P < 0.05; **, P < 0.01; ***, P < 0.001.