DHAV 3CD targets IRF7 and RIG-I proteins to block the type I interferon upstream signaling pathway

Figures

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  Figure 1. Effect of the 3CD protein on the IFNβ signaling pathway.A pCAGGS-3CD-HA and Poly(I:C) were cotransfected into 24-well plate cells (transfection ratio of 1:1), and the cell samples were collected 36 h after transfection and the total RNA was extracted from the samples. Quantitative PCR analysis of the effect of the 3CD protein on poly(I:C)-induced IFN-β and IFN-β-induced MX and OASL mRNA levels. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group. B 0.6 µg pCAGGS-3CD-HA and 0.4 µg pCAGGS-IFNβ-Flag were co-transfected into cells in 12-well plates, and cell samples were collected 36 h after transfection to detect the expression of IFNβ protein.

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  Figure 2. The 3CD protein interacts with IRF7 protein and inhibits the expression of IRF7 protein.A pCAGGS-3CD-HA and pCAGGS-IRF7-Flag were co-transfected or both were separately co-transfected with pCAGGS into 24-well plates (transfection ratio of 1:1), and cell samples were collected 36 h after transfection for indirect immunofluorescence experiments to observe the intracellular localization of the 3CD protein and IRF7 protein. B Cells were grown in 6-well plates and each well was transfected with a total of 1 µg pCAGGS-3CD-HA and 1 µg pCAGGS-IRF7-Flag. Cells were lysed 36 h after transfection, and lysates were immunoprecipitated with mouse anti-Flag or mouse IgG antibodies and subjected to Western blotting. C 0.4 µg pCAGGS-IRF7-Flag was co-transfected into 12-well plates with 0.6 µg pCAGGS-3CD-HA, pCAGGS-3D-HA, pCMV-3 C-HA or empty, respectively, and cell samples were collected 36 h after transfection to detect the expression of IRF7 protein. D Similar transfection and quantitative PCR experiments were performed to analyze the effect of 3CD protein on Poly(I:C)-induced IRF7 mRNA levels as described in Figure 1A. * P < 0.05, ** P < 0.01, *** P < 0.001, compared with the control group. E…

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  Figure 3. Effect of the 3CD protein on the nuclear translocation of the IRF7 protein.A Three experimental groups were set up, both groups I and II were co-transfected with 0.3 µg pCAGGS-IRF7-Flag and 0.5 µg pCAGGS; the third group was transfected with 0.3 µg pCAGGS-IRF7-Flag and 0.5 µg pCAGGS-3CD-HA, and transfect the plasmids into cells in a 24-well plate. 24 h after transfection groups II and III were stimulated with 5 µg/µL Poly (I:C) for 12 h, and cell samples were collected for indirect immunofluorescence experiments. B Images such as those in panel A were subjected to image analysis (see Materials and methods), where > 30 cells for each sample were analyzed to determine Fn/c, the fluorescence in the nucleus relative to that in the cytoplasm. Results are means ± standard errors of the mean (n ≥ 30). C DEFs were cultured in 6-well plates, grouped and transfected as described above for group A (transfection ratio of 3:2), 24 h after transfection groups 2 and 3 were stimulated with 20 µg/µL Poly(I:C) for 12 h, and cell samples were collected for Western blot analysis. D Gray value analysis was performed on the bands in C with ImageJ software to determine the ratio of Pn/c: Pn/c = Pn/Pc,…

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  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…

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  Figure 5. The 3CD protein interacts with the N-terminal domain of RIG-I.A pCAGGS-3CD-HA and pCAGGS-RIG-I (N-terminal)-Flag were co-transfected or both were co-transfected separately with empty plasmid into 24-well plates (transfection ratio of 1:1), and cell samples were collected 36 h after transfection for indirect immunofluorescence assays to observe the intracellular localization of 3CD and RIG-I. B Cells were seeded in 6-well plates, and 1 µg of pCAGGS-3CD-HA and 1 µg of pCAGGS-RIG-I (N-terminal)-Flag were co-transfected into monolayers. Cells were lysed 36 h after transfection, and lysates were immunoprecipitated with mouse anti-Flag or mouse IgG antibodies and subjected to Western blotting.

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  Figure 6. The 3CD protein interferes with the interaction between RIG-I and MAVS. DEFs were co-transfected with 0.8 µg pCAGGS-3CD-HA or empty plasmid, 0.6 µg pCAGGS-3*Flag-MAVS and 0.6 µg pCAGGS-RIG-I (N-terminal)-Myc, respectively, in 6-well plates. Cells were lysed 36 h after transfection and the supernatant was collected for immunoprecipitation assay using an anti-Flag antibody. RIG-I, 3CD, and MAVS were detected by Western blotting using anti-Flag, anti-Myc, and anti-HA antibodies.

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  Figure 7. The 3CD protein reduces RIG-I protein through the proteasome pathway. Cells in 12-well plates were co-transfected with 3CD-HA (0.6 µg) or empty plasmid and RIG-I (N-terminal)-Flag (0.4 µg). At 24 h post-transfection, the broad-spectrum caspase inhibitor Z-VAD-FMK (50 µM), proteasome inhibitor MG132 (20 µM), autophagy inhibitor 3-MA (10 mM), and autophagy-lysosome inhibitor NH4Cl (10 mM) were added to the cell culture medium, while a DMSO negative control group was set up and treated for 12 h. Cell samples were collected after 12 h of treatment, and immunoblotting experiments were performed in the presence or absence of these inhibitors. The expression level of RIG-I was detected. A The effect of MG132 on RIG-I protein degradation. B The effect of 3-MA on RIG-I protein degradation. C The effect of Z-VAD-FMK on RIG-I protein degradation. D The effect of NH4Cl on RIG-I protein degradation.

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  Figure 8. The 3CD protein promotes DHAV-1 replication by inhibiting the RIG-I protein.A Cells in the 24-well plate were transfected with different doses of RIG-I (N-terminal)-Flag (the amount transfected into each group was 0.5 µg, and pCAGGS was used to supplement the insufficient group), and the cells were infected with the DHAV-1 CH strain with MOI = 0.5 for 12 h after transfection. Cell samples were collected 36 h after transfection to detect the viral copy number, and the expression of VP3 and RIG-I was detected by Western blotting. B 0.5 µg RIG-I (N-terminal)-Flag was co-transfected with 0.5 µg 3CD-HA or co-transfected with empty plasmid respectively in 12-well plates, and cells were infected with DHAV-1 CH strain with MOI = 0.5 at 12 h. Cell samples were collected at 36 h after transfection to detect the viral copy number, and the expression of VP3, RIG-I, and 3CD was detected by Western blotting. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared with the control group.

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  Figure 9. A model of DHAV-1-mediated inhibition of IFNβ signaling pathway. After DHAV-1 infection, both viral proteins 3 C and 3CD can inhibit the production of IFNβ by interacting with IRF7 protein and inhibiting the expression of IRF7 protein, and the 3 C protein also inhibits the nuclear translocation of the IRF7 protein, blocking interferon signaling. In addition, the 3CD protein can interact with RIG-I to inhibit its expression, and can also interfere with the formation of a complex between RIG-I and MAVS to ultimately inhibit the RIG-I-mediated signaling pathway.