2B and 3C Proteins of Senecavirus A Antagonize the Antiviral Activity of DDX21 via the Caspase-Dependent Degradation of DDX21

Figure 1. Experimental design for identifying candidate novel cellular factors in mock- and SVA-infected PK-15 cells. (A) PK-15 cells were infected with 0.5 MOI of SVA for 12, 24, 36, 48, 60, or 72 h. The virus-induced CPE was observed and recorded by the microscope. (B) PK-15 cells were infected with 0.5 MOI of SVA for 12, 24, 36, 48, 60, or 72 h, and the viral titers were determined by the TCID50 assay. (C) Schematic illustration of the sample preparation. PK-15 cells mock infected or infected by SVA at an MOI of 0.5 for 48 h were subjected to TMT-based proteomic analysis.

Figure 2. Proteomic analysis of SVA-infected PK-15 cells. PK-15 cells were mock or SVA infected at a MOI of 0.5 and harvested at 48 hpi for proteomic analysis. (A) The Venn diagram represents the number of genes upregulated and downregulated in PK-15 cells in response to SVA infection. (B) Cluster analysis of the upregulated and downregulated genes with the greatest fold change values. Red indicates increased (infected/mock) gene expression, and blue indicates decreased expression. (C, D) Validation of TMT data by qRT-PCR analyses. ZC3H11A, DDX21, IL-18, and ZCCHC9 mRNA levels were upregulated, and STAT1, OAS1, MX2, ZAP, and IFIT1 mRNA levels were downregulated. Values are presented as means ± SD from three independent experiments.

Figure 3. DDX21 affects the replication of SVA. (A) BHK-21 cells were transfected with pCAGEN-DDX21-HA or pCAGEN. At 36 hpt, the cells were inoculated with SVA at an MOI of 0.1. The cells and supernatant were harvested at the indicated times, and the expression level of the VP2 protein of SVA was analyzed by WB with an anti-VP2 pAb. DDX21-HA was detected with an anti-HA mAb. β-Actin served as the internal reference. (B) Virus titers were calculated and expressed as TCID50/ml at the indicated times. (C) BHK-21 cells seeded in 6-well plates were transfected with siRNA-1651, siRNA-673, siRNA-1712, or negative control (NC) (50 pM/well). At 36 hpt, the cells were lysed and analyzed by Western blotting (WB) with an anti-DDX21 polyclonal antibody (mAb), and β-actin was probed as the internal reference. (D) Knocking down the expression of DDX21 promoted the replication of SVA in BHK-21 cells. BHK-21 cells were transfected with NC or siRNA-1651 at 50 pM. At 36 hpt, the cells were infected with SVA at a MOI of 0.1. The cells and the supernatant were harvested at indicated times. The cells were lysed and analyzed by WB using an anti-VP2 pAb. (E) The infectious virus titers in the supernatant were…

Figure 4. SVA degrades endogenous DDX21 during viral infection, and DDX21 is degraded by SVA 2B and 3C. (A) BHK-21 cells in 6-well plates were infected with SVA at a MOI of 0.1, cells were harvested at indicated times, and the expression level of endogenous DDX21 protein was analyzed by WB. (B) HEK293T cells in six-well plates were cotransfected with HA-DDX21 and Flag-VP1, VP2, VP3, 2B, 2C, 3A, 3C, or 3D, cells were harvested at 30 h, and the samples were analyzed by WB. (C, D) HEK293T cells were cotransfected with HA-DDX21 and different concentrations of Flag-2B or Flag-3C for 30 h. The cell lysates were analyzed by WB.

Figure 5. SVA 3C protein targets DDX21 for degradation through its protease activity. (A) HEK293T cells were cotransfected with HA-DDX21 and 3C protein or 3C-H48A for 30 h. The cell lysates were prepared for Western blot analysis using the indicated antibodies. (B) HEK293T cells were cotransfected with HA-DDX21 and 3C protein or 3C-C160A for 30 h. The cell lysates were prepared for Western blot analysis using the indicated antibodies. (C) HEK293T cells were cotransfected with HA-DDX21 and 3C protein or 3C-DM for 30 h. The cell lysates were prepared for Western blot analysis using the indicated antibodies.

Figure 6. DDX21 does not interact with SVA 2B and 3C protein. (A) HEK293T cells were cotransfected with HA-DDX21 and 2B-Flag. (B) HEK293T cells were cotransfected with HA-DDX21 and 3C-flag. Coimmunoprecipitation (Co-IP) was performed using agarose beads conjugated with anti-Flag mAb agarose beads. Precipitated proteins were analyzed by WB using antibodies against the Flag and HA.

Figure 7. DDX21 is degraded through the caspase pathway during SVA infection. Cells were treated with the indicated concentrations of inhibitors for 24 h. MTT method is used to detect BHK-21 cells cytotoxicity. (A) MG132. (B) Z-VAD-FMK. (C) CQ. (D–F) BHK-21 cells in six-well plates were incubated with different inhibitors and then infected with SVA at an MOI of 0.1. The cell lysates were analyzed by WB. MG132 was added at 10 to 20 µM, Z-VAD-FMK was added at 10 to 20 µM, and CQ was added at 50 to 100 µM. (G–I) BHK-21 cells were cotransfected with HA-DDX21 and 2B-Flag and then treated with different inhibitors. MG132 was added at 10 to 20 µM, Z-VAD-FMK was added at 10 to 20 µM, and CQ was added at 50 to 100 µM. The cell lysates were analyzed by WB. (J–L) BHK-21 cells were cotransfected with HA-DDX21 and 3C-Flag and then treated with different inhibitors. MG132 was added at 10 to 20 µM, Z-VAD-FMK was added at 10 to 20 µM, and CQ was added at 50 to 100 µM. The cell lysates were analyzed by WB. The statistical significance of differences was determined using a Student’s t-test (NS, not significant; ** p < 0.01; *** p < 0.001).