Figure 4

The 3CD protein induces the reduction of RIG-I and inhibits RIG-I-mediated signal transduction.A Cells were grown in 12-well dishes, and pCAGGS-RIG-I (N-terminal)-Flag, pCAGGS-3*Flag-MDA5, pCAGGS-3*Flag-MAVS, pCAGGS-TBK1-Flag were transfected with pCAGGS-3CD-HA or pCAGGS, respectively (transfection ratio of 2:3) and cell samples were collected for Western blot analysis at 36 h of transfection. B Similar transfection and quantitative PCR experiments were performed to analyze the effect of 3CD protein on Poly(I:C)-induced RIG-I mRNA levels as described in panel A of Figure 1. NS, not significant, compared with the control group. C DEFs were inoculated in 12-well plates and 0.4 µg of pCAGGS-RIG-I(N-terminal)-Flag was co-transfected with different doses of pCAGGS-3CD-HA (the amount transfected into each group was 1 µg, and pCAGGS was used to supplement the insufficient group). Cell samples were collected 36 h post-transfection and assayed for 3CD and RIG-I protein expression. D Cells in 12-well plates were co-transfected with plasmids expressing 3CD-HA (0.6 µg) or empty plasmid and RIG-I (N-terminal)-Flag (0.4 µg). Cells were collected at 12, 24, 36, and 48 h, and cell lysates were analyzed by Western blotting to detect the expression levels of RIG-I and 3CD. E pCAGGS-3CD-HA or pCAGGS was co-transfected with pCAGGS-RIG-I(N-terminal)-Flag, IFNβ pro-Luc, and the reference plasmid pRL-TK into 24-well plates (the transfection ratio of the first three plasmids was 2:1:1, and the amount of pRL-TK transfected was 1/20 that of IFN-β pro-Luc). 36 h after transfection, cell samples were collected. The dual-luciferase reporter system was used to detect IFN-β promoter activity. ** P < 0.01, ***P < 0.001, compared with control group. F 0.4 µg of pCAGGS-RIG-I (N-terminal)-Flag was transfected with 0.6 µg of pCAGGS-3CD-HA, pCAGGS-3D-HA, pCMV-3 C-HA or pCAGGS into cells in 12-well plates respectively, and cell samples were collected 36 h after transfection to analyze the expression of each protein.