FIGURE 2.

Rad51 is not required for HCV replication. (A) Huh7 cells harboring HCV subgenomic replicon derived from genotype 1b were transfected with the indicated siRNAs. At 48 h or 72 h after transfection, total cell lysates were immunoblotted with the indicated antibodies. HCV RNA and Rad51 mRNA levels were analyzed by qRT-PCR. (B) Huh6 cells harboring HCV subgenomic replicon derived from genotype 2a were transfected with the indicated siRNAs. At 72 h after transfection, total cell lysates were immunoblotted with the indicated antibodies. HCV RNA and Rad51 mRNA levels were analyzed by qRT-PCR. (C,D) Huh7.5 cells were infected with Jc1 (MOI = 1) for 4 h. At 48 h post-infection, cells were transfected with the indicated siRNAs. At 48 h after transfection, protein expression levels were determined by immunoblot analysis using the indicated antibodies (C) and intracellular RNA levels were analyzed by qRT-PCR (D). Intracellular RNA levels were normalized to levels of β-actin mRNA. (E) Schematic illustration of the experimental design. (F) Huh7.5 cells infected with Jc1 (MOI = 1) were transfected with the indicated siRNAs for 24 h and then supplied with fresh media. The culture supernatants were harvested at the indicated time points and extracellular HCV RNA levels were analyzed by qRT-PCR. (G) Supernatants harvested at the various time points were used to infect naïve Huh7.5 cells and viral infectivity was determined by qRT-PCR. The asterisk indicates significant difference (^∗p < 0.05) from the value for the negative siRNA control. Experiments were carried out in triplicate. Error bars indicate standard deviations. (H) Huh7.5 cells were transfected with the negative siRNA, Rad51-specific siRNA, and CD81-specific siRNA as a positive control, and then infected with HCV. At 12 h post-infection, HCV RNA levels were quantified by qRT-PCR. The asterisks indicate significant differences (^∗p < 0.05, ^∗∗p < 0.01) from the value for the negative siRNA control. Error bars indicate standard deviations.