Figure 1: ARFRP1 is required for HCV propagation.

(A) Huh7.5 cells were either mock-infected or infected with HCV Jc1 for 4 h. Total cellular lysates harvested at the indicated time intervals were immunoblotted with the indicated antibodies. (B) Huh7.5 cells were transfected with 20 nM of the indicated siRNAs. At 2 days after siRNA transfection, cells were infected with Jc1 for 4 h. At 48 h postinfection, intracellular RNA levels of both HCV and ARFRP1 were quantified by qRT-PCR. Negative, scrambled siRNA; positive, HCV-specific siRNA. The asterisk indicates significant difference (*p < 0.05) from the value for the negative control. (C) Huh7.5 cells transfected with the indicated siRNAs were infected with Jc1. Total cell lysates harvested at 48 h postinfection were immunoblotted with the indicated antibodies. (D) Extracellular RNAs isolated from the culture supernatant were quantified by qRT-PCR. (E) Huh7.5 cells were transfected with 50 nM of the indicated siRNAs for 96 h and then cell viability was assessed by MTT assay. (F) Naïve Huh7.5 cells were infected with virus-containing culture supernatants harvested from (D). HCV infectivity was determined by a focus-forming assay (upper panel) and total cell lysates were immunoblotted with the indicated antibodies (lower panel). (G) HCV subgenomic replicon cells were transfected with the indicated siRNAs. At 72 h after siRNA transfection, intracellular RNA levels of both HCV and ARFRP1 were quantified by qRT-PCR. The asterisks indicate significant differences (*p < 0.05; **p < 0.01) from the value for the negative control. (H) HCV subgenomic replicon cells were transfected with the indicated siRNA constructs. Total cell lysates harvested at 72 h after transfection were immunoblotted with the indicated antibodies. The band intensity was quantified using ImageJ software. (I) Huh7.5 cells transfected with the indicated siRNAs were infected with Jc1 for 4 h and then further transfected with the indicated combinations of plasmids. At 48 h after transfection, cell lysates were immunoblotted with the indicated antibodies. (J) Huh7.5 cells were treated as described in legend to Fig. 1H and then extracellular RNAs were quantified by qRT-PCR. Flag-ARFRP1-WT, Flag-tagged wild-type ARFRP1; Flag-ARFRP1-SR, Flag-tagged siRNA-resistant mutant ARFRP1. All experiments were performed in duplicate.