Silencing of PC impairs production of infectious HCV.

(A) Knockdown of PC had no effect on viral protein levels in HCV replicon cells. Huh7 cells harboring HCV replicon were transfected with 10 nM of negative (Neg), positive (Pos), or the indicated siRNA duplexes for 3 days. Total RNAs were extracted and intracellular HCV RNA was analyzed by qRT-PCR. Negative, irrelevant siRNA pool; positive, HCV-specific siRNAs. (B) Total replicon cell lysates harvested at 3 days after siRNA transfection were immunoblotted with the indicated antibodies. (C) Huh7.5 cells were transfected with 10 nM of the indicated siRNA. At 2 days after siRNA transfection, cells were infected with Jc1 for 4 h. At 48 h postinfection, cell proliferation was assessed by the MTT assay. Huh7.5 cells were treated as described in (C). At 48 h postinfection, both intracellular HCV RNA (D) and extracellular HCV RNA isolated from culture supernatants (F) were analyzed by qRT-PCR. (E) Total cell lysates harvested at 48 h after Jc1 infection were immunoblotted with the indicated antibodies. (G) Intracellular infectious HCV particles were prepared by 4 rounds of freeze and thaw treatments from cells treated as in figure legend to C. Neg indicates cells infected with intracellular HCV isolated from the Negative siRNA-transfected cells. PC denotes cells infected with intracellular HCV isolated from the PC siRNA-transfected cells. Pos denotes cells infected with intracellular HCV isolated from the Positive siRNA-transfected cells. (H) Extracellular infectious HCV particles were prepared from the culture media in cells treated as in figure legend to C. Neg indicates cells infected with extracellular HCV isolated from the Negative siRNA-transfected cells. PC denotes cells infected with extracellular HCV isolated from the PC siRNA-transfected cells. Pos denotes cells infected with extracellular HCV isolated from the Positive siRNA-transfected cells. (G, H) Naïve Huh7.5 cells were then infected with intracellular infectious HCV (G) and extracellular infectious HCV (H). Total cell lysates harvested at 2 days postinfection were immunoblotted to determine the indicated protein levels, and HCV RNA levels were analyzed by qRT-PCR (top panels). Naïve Huh7.5 cells treated as described above were analyzed for immunofluorescence using anti-NS5A antibody (bottom panels). Cells were counterstained with DAPI to label nuclei. Samples were analyzed for immunofluorescence staining using a Zeiss LSM 700 laser confocal microscopy system. Asterisks indicate significant differences (*, P<0.05, **, P<0.01) from the value for the negative control. Error bars indicate standard deviations.