FIG. 4.

GBF1 is the brefeldin A-sensitive factor required for HCV replication. (A) Huh-7 cells were transfected with the indicated siRNAs and infected with HCV. Luciferase activity was measured at 24 h postinfection. The luciferase activity from control siRNA-transfected cells (si control) is expressed as 100%. Error bars indicate standard errors of the means for 6 experiments. (B) siRNA-mediated depletion of target proteins was verified by immunoblot analysis. (C) Huh-7 cells were infected with HCV-RLuc or GFP-expressing adenovirus in the presence of 1 μg/ml BFA, 0.02% ethanol (BFA stock solvent), 10 μM golgicide A (GCA), or 0.02% dimethyl sulfoxide (DMSO) (golgicide A stock solvent). Both drugs were present for 8 h. At 24 h postinfection, cells were harvested for luciferase assay (HCVcc) or fluorescence-activated cell sorter analysis (adenovirus). The luciferase activity and number of adenovirus-infected cells from ethanol- or DMSO-treated samples are expressed as 100%. (D) Huh-7 cells harboring a subgenomic replicon were transfected with the indicated siRNAs. Cells were lysed, and cell lysates were analyzed by immunoblotting with antibodies to NS5A and actin. (E) Huh-7 cells were transfected with expression plasmids for GBF1, BFA-resistant mutant GBF1-M832L, GBF1 inactive mutant E794K, or YFP. Transfected cells were infected with HCVcc and cultured in the presence or absence of BFA. Cells were fixed at 24 h postinfection and processed for immunofluorescence detection of E1. Results are presented as percentage of infected cells. (F) Huh-7 cells were transfected with expression plasmids for GBF1-M832L or GBF1-E794K, infected with HCV-RLuc in the presence of 0.2% ethanol or the indicated concentration of BFA, and cultured in the presence of BFA for 8 h and then in the absence of the drug. Luciferase activity was measured at 24 h postinfection. The luciferase activity from ethanol-treated cells is expressed as 100%.