African Swine Fever Virus MGF360-14L Negatively Regulates Type I Interferon Signaling by Targeting IRF3

Abstract

African swine fever (ASF) is a devastating infectious disease caused by African swine fever virus (ASFV). The ASFV genome encodes multiple structural and non-structural proteins that contribute to evasion of host immunity. In this study, we determined that the viral non-structural protein MGF360-14L inhibits interferon-β (IFN-β) promoter activity induced by cGAS-STING signaling. MGF360-14L was also found to downregulate expression of the IRF3 protein and promote its degradation through ubiquitin-meditated proteolysis. Moreover, MGF360-14L was shown to interact with and destabilize IRF3 by facilitating E3 ligase TRIM21-mediated K63-linked ubiquitination of IRF3. Overall, our study revealed that MGF360-14L promotes degradation of IRF3 through TRIM21, thereby inhibiting type I interferon production. These findings provide new insights into the mechanisms underlying ASFV immune evasion.

Keywords: African swine fever virus; IRF3; immune evasion; interferon; ubiquitination.

Figure 1

MGF360-14L inhibited IFN-β mRNA production and IFN-β promoter activity. PK-15 (A) and HEK293T (B) cells were transfected with MGF360-14L plasmids and 5 μg/mL of poly(I:C) for 24 h and then harvested for qRT-PCR assay to determine IFN-β mRNA levels. MGF360-14L inhibited cGAS/STING-mediated activation of IFN-β (C), and the inhibitory effect was dose-dependent (D). HEK293T cells were co-transfected with IFN-β-luc (20 ng), pRL-TK (2 ng), cGAS-HA (10 ng), STING-HA (40 ng), or the pcDNA3.1-HA empty vector (50 ng) and MGF360-14L-Flag (0 ng, 50 ng, 100 ng, 200 ng). Luciferase activity was detected at 24 h post-transfection. Expression of cGAS-HA, STING-HA, and MGF360-14L-Flag was analyzed by western blotting. (E, F) MGF360-14L inhibited cGAS/STING-mediated mRNA and protein levels of IFN-β. HEK293T cells were co-transfected with cGAS-HA (100 ng), STING-HA (100 ng), or the pcDNA3.1-HA empty vector (100 ng) and MGF360-14L-Flag (0 ng, 50 ng, 100 ng, 200 ng). The mRNA level of interferon-β in cell lysate was detected by qPCR, and the protein expression level of interferon-β in supernatant was detected by ELISA. Data shown are the mean ± SEM; *p < 0.001, **p < 0.001, ***p < 0.001. Representative results are from at least three independent experiments.

Figure 2

MGF360-14L inhibits IRF3-mediated IFN-β promoter activation. (A, B) HEK293T cells were co-transfected with IFN-β-luc (20 ng), pRL-TK (2 ng), TBK1 (50 ng) or IRF3-5D (50 ng), and MGF360-14L-Flag (50 ng) or empty plasmid. At 24 h post-transfection, cells were treated and analyzed using dual-luciferase reporter assays. (C) HEK293T cells were transfected with MGF360-14L-Flag (0 ng, 200 ng, 400 ng and 600 ng) or empty vector. (D) HEK293T cells were transfected with IRF3-HA (200 ng) and MGF360-14L-Flag (0 ng, 200 ng, 400 ng, and 600 ng) or empty vector. (E) HEK293T cells transfected with MGF360-14L-eGFP and then treated with DMSO or MG132 (25 μM), CQ (50 μM), 3-MA (10 mM) and Z-VAD-FMK (20 μM) for 6 h. Expression of IRF3, IRF3-HA, TBK1-HA, IRF3-5D-HA, MGF360-14L-Flag and GAPDH were assessed by western blot analysis. Data shown are the mean ± SEM; **p < 0.001, ***p < 0.001. Representative results are from at least three independent experiments.

Figure 3

MGF360-14L interacts with TRIM21. (A) IP samples were separated by SDS-PAGE and stained with Coomassie blue; exogenously expressed MGF360-14L is indicated by the red box. (B) MS analysis of MGF360-14L-associated proteins, following by Gene Ontology (GO) analysis. High-scoring peptides were listed in the table. (C) HEK293T cells were transfected with MGF360-14L-Flag and TRIM21-HA plasmids. (D) HEK293T cells were transfected with MGF360-14L-Flag plasmids for 18 h and then incubated with SeV (1MOI) for 6 h. The cells were lysed and subjected to IP using anti-Flag, anti-HA monoclonal antibodies or control IgG. Precipitates were analyzed by western blot. (E) PK-15 cells were transfected with MGF360-14L-Flag and TRIM21-HA plasmids. At 24 h after transfection, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton™ X-100, blocked with 5% BSA, and then treated with primary antibody and secondary antibody. Finally, laser confocal microscopy was performed after DAPI nuclear staining.

Figure 4

MGF360-14L destabilizes IRF3. (A, B) HEK293T cells were co-transfected with IFN-β-luc (20 ng), pRL-TK (2 ng), cGAS (10 ng), STING (40 ng) or IRF3-5D-HA (50 ng), MGF360-14L-Flag (50 ng), and TRIM21-HA (50 ng) or empty plasmid. At 24 h post-transfection, cells were treated and analyzed using dual-luciferase reporter assays. (C) HEK293T cells were transfected with MGF360-14L-Flag (500 ng), TRIM21-Myc (500 ng), IRF3-HA (500 ng) or empty plasmids. (D) HEK293Tcells were transfected with MGF360-14L-Flag (500 ng), sh-TRIM21 (500 ng,1000 ng) or empty plasmid. (E) HEK293T cells were transfected with MGF360-14L-eGFP (or MGF360-14L-Flag), TRIM21-Myc and IRF3-Flag (or IRF3-HA) plasmids. (F) HEK293T cells were transfected with MGF360-14L-Flag plasmids for 18h and then incubated with SeV (1MOI) for 6 h. The cells were lysed, and subjected to IP using anti-Flag or control IgG. Precipitates were analyzed by WB. (G, H) IP with anti-Flag beads analysis of lysates of HEK293T cells transfected with Ub-HA, or Ub-K48-HA, or Ub-K63-HA, TRIM21-Myc, MGF360-14L-eGFP and IRF3-Flag for 24h. (I) IP with anti-IRF3 beads analysis of lysates of HEK293T cells transfected with Ub-HA, or Ub-K63-HA, shTRIM21 and MGF360-14L-eGFP for 24h. Expression of MGF360-14L-Flag (or MGF360-14L-eGFP), TRM21-Myc (or TRIM21-HA), IRF3-HA (or IRF3-Flag),Ub-HA, Ub-K48-HA, Ub-K63-HA and GAPDH were assessed by Western blot analysis. Data shown are the mean ± SEM; *p < 0.001, **p < 0.001. Representative results are from at least three independent experiments.