Fig. 4.

The exonuclease-deficient ISG20 mutant inhibits the antiviral action of IFN against intracellular HCV RNA replication. (A) 7.5-Bsr and 7.5-ISG20m cells were mock-treated or pretreated with 1000 U/mL of recombinant human IFN-α for 18 h. IFN was then removed and cells transfected with SGRm-JFH1RL replicon RNA. Replication kinetics was analyzed the same way as in Fig. 2A. Data shown were representative of three independent experiments. (B) Triplicate wells of 7.5-Bsr and 7.5-ISG20m cells were pretreated with 0, 5, 10, 25 and 100 U/mL of IFN-α for 24 h. After removal of IFN, cells were infected with JFH1 virus at an MOI = 0.05. Cell free culture supernatants were harvested at 48 h postinfection and infectious virus titer determined by fluorescent focus forming assay. Data are presented as percentage of virus titer relative to cells treated with 0 U/mL IFN. Data are representative of 2 independently conducted experiments. (C) 7.5-Bsr and 7.5-ISG20m cells respond similarly to IFN. Cells were treated with 0, 10, 50, 100, and 1000 U/mL of IFN-α for 7 h prior to total RNA extraction and realtime PCR analysis of ISG56 and MxA mRNA expression. Fold change of each gene was calculated by normalizing the data to 7.5-Bsr mock treated cells (there was no difference in the baselines of ISG56 and MxA mRNAs between mock-treated 7.5-bsr and 7.5-ISG20m cells).