FIG. 3.
Long-term effect of esiRNAs on the replication of a subgenomic HCV replicon. The effects of single (A) and consecutive (B) esiRNA transfections on replicon-dependent luciferase reporter gene expression are shown. About 2 × 105 cells of the Huh-7 clone 9B (containing the HCV 1b replicon I389/NS3-3′/LucUbiNeo-ET) were transfected with 1 μg of HCV genotype 1b-specific esiRNAs (ORF1 and ORF3) or unrelated esiRNAs (EGFP) or were left untreated. At the times indicated, cells were lysed and luciferase activities were determined, at least in duplicate. Arrows and dotted lines indicate times of transfection and cell splitting (ratio of 1:3), respectively. RLU, relative light units. (C) Effect of genotype 1b-specific esiRNA transfections on the replicon copy number. RNAs were prepared from cells that had been lysed 11 days after the first esiRNA transfection, and the numbers of replicon molecules were determined by quantitative RT-PCR. The concentrations of viral RNAs are given in replicon copy numbers per microgram of total RNA. Hatched columns, viral RNA concentration in control cells; black columns, viral RNA concentration after transfection of HCV-specific esiRNAs. The figure shows the results from a single representative experiment.