Figure 8

Stable expression of IFNAR1 improves the antiviral response of IFN-α in resistant R-17/3 cells. Cured Huh-7 cells prepared from S-5/15 and R-17/3 cells were transfected with full-length JFH1-GFP chimeric HCV RNA by electroporation. After 72 hours, these cells were treated with IFN-α (1000 IU/ml) for an additional 72 hours. Replication of HCV in the transfected cells was examined by GFP expression and positive-strand HCV-RNA was detected by RPA using a probe targeted to the highly conserved 5'UTR. (A) HCV RNA levels before and after IFN-α treatment was measured by RPA. Left panel shows that high level HCV RNA replication in different Huh-7 clones without IFN-α treatment. Right panel shows the HCV RNA level in the same set of Huh-7 cell lines after IFN-α treatment. IFN-α inhibits the level of HCV RNA in IFN-α sensitive Huh-7 cells, but not in the resistant Huh-7 cell line. The antiviral effect of IFN-α can be restored upon stable expression of IFNAR1 in the resistant Huh-7 cell line. The GAPDH mRNA levels served as controls. (B) Expression and replication of HCV-GFP in the transfected Huh-7 cells before and after IFN-α treatment was measured by fluorescence microscopy at 20X magnification. The upper panel shows the expression HCV-GFP chimera protein in different Huh-7 cell lines transfected with HCV without IFN-α treatment. The lower panel shows the expression HCV-GFP in the same set of Huh-7 cells after IFN-α treatment. Positive expression indicates the impaired antiviral effect of IFN-α against HCV.