Figure 2.

The m⁶A modification of HCV genome inhibits IRF-3–mediated IFN signals. A–C, Huh7 cells treated with siMETTL3 + 14 or control siRNA were transfected with JFH-1 GND RNA. IFN-β mRNA levels were assessed by qRT-PCR at 16 h post–JFH-1 GND RNA transfection. The HCV genome titers were analyzed by qRT-PCR for normalizing IFN-β mRNA (B). The p-IRF-3 protein levels were analyzed by immunoblotting in whole-cell lysates (C). D–F, FLAG-METLL3/14 transfected with Huh7 cells were treated with JFH-1 GND RNA for 16 h. The cells were harvested to assess expression levels of IFN-β mRNA (D) or the indicated proteins (F). The input HCV genome levels were quantified for calculating IFN-β mRNA (E). G, schematics indicate the location of the A8766C mutation of the m⁶A site in the NS5B-3′-UTR region of the HCV genome. The A331C mutation is in domain IV of the HCV IRES region. Red indicates m⁶A peak by MeRIP analysis. Yellow indicates the DRACH motif in the m⁶A peak. H–J, qRT-PCR analysis of IFN-β mRNA relative to input HCV genome in JFH-1 GND RNA or JFH-1 GND A8766C RNA transfected with Huh7 cells. The cells were harvested at 16 h post-transfection, and the relative levels of IFN-β mRNA (H) or p-IRF-3 (J) were analyzed. The input JFH-1 GND RNA levels were analyzed by qRT-PCR (I). JFH-1 A331C RNA was used as a control. The data for this figure are from three independent experiments, and the bars represent the means ± S.D. Statistical significance of the difference between groups was determined via an unpaired Student's t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Ctrl, control.