Figure 3

RO8191 requires and binds IFNAR2.

(a, b) The anti-HCV replicon activity of RO8191 was attenuated by knockdown of IFNAR2 (b), but not IFNAR1 (a). Inhibition of HCV replicon replication by each agent is shown (the mean and SD from 3 experiments). The HCV replicon cells were transfected with 50 nM of the indicated siRNAs (blue, red and yellow bars). Forty-eight hours after transfection, the HCV replicon cells were treated with 1.5 μM RO8191 or 3 IU/mL IFN-α for 24 h. Twenty-four hours after treatment with each agent, the replication levels of HCV RNA were analyzed using a luciferase assay. (c, d) U5A cells that lack IFNAR2 were transfected with either an empty vector or a vector expressing the IFNAR2 gene. (c) Forty-eight hours after transfection, the cells were treated with 50 μM RO8191 (red bars) or 100 IU/mL IFN-α (yellow bars). After an additional 8 h of incubation, total RNA was extracted from the U5A cells and the OAS1 mRNA level was measured using real-time RT-PCR. The values shown are relative to the mRNA level of human β-actin. (d) Forty-eight hours after transfection, the cells were lysed and the whole cell lysates were immunoblotted with the indicated antibodies. (e) Real-time kinetic SPR analysis of the binding of RO8191 to the IFNAR2 ECD (red and blue lines). The results are consistent with 1:1 binding. PEG-IFN-α2a was also injected as a positive interacting control for IFNAR2 (black line, K_D: 30 nM). (f, g) The phosphorylation of STAT1 was attenuated by a knockdown of IFNAR2 (g) but not IFNAR1 (f). The HCV replicon cells were transfected with the indicated siRNAs (10 nM). Forty-eight hours after transfection, the cells were treated for 15 min with 10 μM RO8191 or 200 IU/mL IFN-α. The total lysates were subjected to western blot analysis to analyze the phosphorylated and total protein levels of STAT1. The data were statistically analyzed using Student’s t-test.