FIG 7.

RAD51AP1 is required for the assembly step of the HCV life cycle. (A to D) Huh7.5 cells infected with Jc1 were transfected with the indicated siRNAs. At 48 h posttransfection, intracellular RNA levels (A), protein levels (B), and extracellular HCV RNA levels (C) were analyzed by qRT-PCR. (D) Naive Huh7.5 cells were infected with HCV-containing culture supernatant harvested from the cells for panel A, and viral infectivity was determined by measuring intracellular HCV RNA (left) and protein (right) levels. (E) Huh7.5 cells were transfected with the indicated siRNAs. At 96 h posttransfection, cell viability was determined by WST assay. (F) Huh7.5 cells infected with Jc1 were transfected with the indicated siRNAs. At 48 h posttransfection, cells were lysed by repetitive cycles of freezing and thawing. Intracellular infectivity was determined by limiting-dilution assay (TCID₅₀). (G) Huh7.5 cells infected with Jc1 were transfected with the indicated siRNAs. At 48 h posttransfection, supernatants were collected and extracellular infectivity was determined by limiting-dilution assay (TCID₅₀). (H) Percentages of intracellular and extracellular infectivity relative to the total were determined. (I) (Top) Huh7.5 cells infected with Jc1 were transfected with the indicated siRNAs. At 24 h post-siRNA transfection, cells were further transfected with either wild-type or siRNA-resistant RAD51AP1 plasmid. At 48 h post-plasmid transfection, cell lysates were immunoblotted using the indicated antibodies. (Bottom) Naive Huh7.5 cells were infected with HCV-containing culture supernatant harvested from the cells described for the top panel. At 48 h postinfection, protein expression was analyzed by immunoblot analysis using the indicated antibodies. (J) (Top) Huh7.5 cells infected with Jc1 were transfected with either empty vector or Flag-tagged RAD51AP1 plasmid. At 2 days postinfection, cell lysates were subjected to immunoblot analysis using the indicated antibodies. (Bottom) Naive Huh7.5 cells were infected with Jc1-containing culture supernatant harvested from the cells described for the top panel. At 48 h postinfection, protein expression was analyzed by immunoblot analysis using the indicated antibodies. (K) Huh7.5 cells were infected with Jc1. At 72 h postinfection, cells were transfected with either negative-control or RAD51AP1 siRNA. At 48 h posttransfection, total cell lysates were immunoprecipitated with either NS5A antibody (top panels) or NS3 antibody (bottom panels). Bound HCV RNAs were analyzed by qRT-PCR. (L) Huh7.5 cells infected with Jc1 were further transfected with the indicated siRNAs. At 48 h posttransfection, the cells were lysed by three repeated cycles of freezing and thawing and subjected to RNase protection assay. As a positive control, samples were treated with proteinase K (PK) prior to RNase treatment to remove protecting capsids. (Top) Input RNA amounts of both HCV and RAD51AP1 before RNase protection assay were quantified by qRT-PCR. (Bottom) The amounts of residual HCV RNA were analyzed by qRT-PCR. Experiments were performed in triplicate. The asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; and ns, not significant).