Porcine Sapelovirus 3Cpro Inhibits the Production of Type I Interferon

Figure 1. PSV infection inhibits host type I IFN production. (A) HEK293T cells were seeded in 12-well plates, and the monolayer cells were pretreated with IFN-β at the indicated concentration for 12 h. Then the cells were infected with VSV-GFP (0.1 MOI) or SVV (0.01 MOI) for 24 h. The CPE was observed in microscopy. Virus titer was determined by plaque assay. (B) HEK293T cells were seeded in twenty-four-well plates, and the monolayer cells were co-transfected with 100 ng IFN-β reporter and 5 ng pRL-TK (as an internal control) plasmids for 20 h. Then the cells were infected with Sev (25 HA) or PSV (0.1 MOI) or not for the indicated times. The cells were then subjected to dual-luciferase assays at 12 h postinfection (hpi). (C) HEK293T cells were seeded in 24-well plates, and the monolayer cells were infected with Sev (25 HA) or PSV (0.1 MOI) or not for the indicated times. The mRNA expression level of IFN-β was evaluated with qPCR assay, and housekeeping gene GAPDH was used as the control. (D) PK15 cells were seeded in 24-well plates, and the monolayer cells were co-transfected with 200 ng IFN-β reporter and 5 ng pRL-TK (as an internal control) plasmids for 20 h. Then the cells were infected…

Figure 2. PSV 3Cpro suppresses Sev-induced type I IFN production. (A) HEK293T cells were seeded in 24-well plates, and the monolayer cells were co-transfected with 300-ng plasmids expressing indicated PSV viral protein or empty plasmids and 100 ng IFNβ-Luc along with 5 ng pRL-TK for 20 h, and then the cells were infected or uninfected with Sev (25 HA) for 12 h. The cells were then subjected to dual-luciferase assays. PSV viral protein expression was detected by Western blotting. (B) HEK293T cells were seeded in 24-well plates, and the monolayer cells were transfected with 300-ng empty plasmids, GST-L, HA-3C, or HA-3D for 20 h, and then the cells were infected with Sev (25 HA) or not. The mRNA expression level of IFN-β was measured by qPCR assay, and housekeeping gene GAPDH was used as the control. L, 3Cpro, or 3D expression was detected by Western blotting. (C) HEK293T cells were seeded in 12-well plates, and the monolayer cells were transfected with 500 ng GST-L, HA-3C, and HA-3D for 20 h, and then the cells were infected with Sev (25 HA) or not. The cells were collected for Western blotting. (D) HEK293T cells were co-transfected with different doses of 3Cpro-expressing plasmids (0 ng, 50…

Figure 3. PSV 3Cpro suppresses Sev-induced type I IFN production dependent on its protease activity of 3C. (A) Amino acid sequence alignment results of 3Cpro derived from PSV (MT080999), SVV (APA28975), FMDV (NP74046), EMCV (NP740410), HAV (AKI05745), and EV71 (AFP66570). (B) Schematic diagram of PSV 3Cpro and its mutants. (C) HEK293T cells were transfected with 500 ng HA-3C or its mutants for 20 h, and then the cells were infected or not with Sev (25 HA) for 9 h. The cells were collected for Western blotting.

Figure 4. PSV 3Cpro inhibits IFN-β activation by downregulation of MDA5/TBK1 and cleavage of MAVS. (A) HEK293T cells were seeded in 24-well plates, and the monolayer cells were co-transfected with 300 ng indicated adaptor-expressing plasmids and 300 ng of empty vector or a plasmid encoding HA-3C for 24 h. The cells were collected for Western blotting. (B,C) HEK293T cells were co-transfected with different doses of HA-3C (0 ng, 200 ng, 800 ng), and 500 ng indicated adaptor-expressing plasmids. The cells were collected for Western blotting. (D) HEK293T cells were transfected with different doses of HA-3C (0.5 μg, 1 μg, 2 μg) for 24 h. The cells were collected for Western blotting. (E) HEK293T cells were infected with 0.5 MOI PSV for the indicated times, and then the cells were collected for Western blotting. Data are represented as means ± SD.

Figure 5. PSV 3Cpro inhibits the transcription level of MDA5 and TBK1. (A,B) HEK293T cells were seeded in 12-well plates, and the monolayer cells were co-transfected with 500 ng Flag-MDA5 or Flag-TBK1 and HA-3C for 12 h. Then the cells were treated with DMSO, MG132, NH4Cl, and Z-VAD-FMK for 12 h. The cells were collected for Western blotting. (C,D) The monolayer HEK293T cells in 12-well plates were co-transfected with 500 ng Flag-MDA5 or Flag-TBK1 and HA-3C or HA-3C-mutant. At 24 h post-transfection (hpt), the cells were collected and lysed for Western blotting. (E,F) The monolayer HEK293T cells in 12-well plates were co-transfected with different doses of HA-3C (0 ng, 200 ng, 800 ng) for 24 h, and then the cells were collected for RT-qPCR. 3Cpro expression was detected by Western blotting using the HA antibody. Student’s t-test: **P < 0.01, ***P < 0.001.

Figure 6. PSV 3Cpro cleaves MAVS through the apoptosis pathway. (A) HEK293T cells were seeded in 12-well plates and co-transfected with 500 ng Flag-MAVS and HA-3C for 12 h, then the cells were treated with DMSO, MG132, NH4Cl, and Z-VAD-FMK for 12 h. The cells were then collected for Western blotting. (B) The monolayer HEK293T cells in 12-well plates were co-transfected with 500 ng Flag-MAVS and HA-3C or HA-3C-mutant. At 24 hpt, the cells were collected and lysed for Western blotting. (C) HEK293T cells were infected with 0.5 MOI PSV for the indicated times, and then the cells were collected and lysed for Western blotting. (D) HEK293T cells were transfected with different doses of HA-3C (0.5 μg, 1 μg, 2 μg) for 24 h, and then the cells were collected and lysed for Western blotting. (E) HEK293T cells were transfected with 500 ng HA-3C or its mutant for 12 h. The Z-VAD-FMK (50 μM) were added and maintained in the cells for another 12 h, and then the cells were collected and lysed for Western blotting.

Figure 7. PSV 3Cpro cleaves MAVS at D429. (A) Schematic diagram of MAVS and its mutants. (B) HEK293T cells were co-transfected with 500 ng HA-3C and Flag-MAVS or its mutants for 24 h. Cell lysates were analyzed by Western blotting. (C, D) HEK293T cells were transfected with 300 ng MAVS or MAVS (D429A) and HA-3C (WT) or HA-3C (DM). Luciferase activity assay assessed IFN-β promoter activity and qPCR tested the mRNA expression level of IFNβ. (E, F) The effects of cleavage fragments of MAVS by PSV 3Cpro on MAVS-mediated IFN-β production were assessed via luciferase activity assay and qPCR assay. The expression of MAVS and its mutants was detected by Western blotting using the FLAG antibody. Data are represented as means ± SD. Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001.