Fig 1.
ASFV infection induces low levels of IL-1β and type I IFN in PAMs.
(A-E) PAMs were either mock-infected or infected with ASFV at an MOI of 0.01, 0.1, and 1.0, respectively. At 24 hpi, the IL-1β (A), TNF-α (B), IL-6 (C), IFN-α (D) and IFN-β (E) levels in the cell culture supernatants were detected by ELISA and the mRNA levels in the cell lysates were determined by qPCR. PAMs were infected with HSV-1 at an MOI of 1.0 as a control. The genome copy numbers of ASFV in PAMs were measured by qPCR (F). A p value of less than 0.05 was considered statistically significant. *p<0.05, **p<0.01, ***p<0.001.
Fig 2.
ASFV infection inhibits inducer-mediated IL-1β and IFN-β production in PAMs.
(A-D) PAMs were either mock-infected or infected with ASFV at an MOI of 0.01, 0.1, and 1.0, respectively. At 24 hpi, PAMs were treated with LPS (100 ng/ml) for 8 h and then stimulated with Nigericin (5 μM) for another 4 h (A), or with LPS (100 ng/ml) and poly(dA:dT) (5 μg/ml) for 12 h (B), or with SeV (1 MOI) (C) or poly(dA:dT) (5 μg/ml) (D) for 12 h, then the secretion and the mRNA expression levels of IL-1β were detected by ELISA and qPCR, respectively. A p value of less than 0.05 was considered statistically significant. *p<0.05, **p<0.01, ***p<0.001.
Fig 3.
Screening for ASFV-encoded MGFs that inhibit IL-1β and IFN-β production.
(A) HEK293T cells were transfected with a plasmid expressing one of the ASFV-encoded pMGFs in the presence of the iGLuc-based NLRP3 inflammasome system (100 ng iGLuc, 10 ng pCAGGS-caspase-1, 10 ng pCAGGS-ASC, and 12.5 ng pCAGGS-NLRP3), and the supernatants were assessed for luciferase activity at 24 hpt. (B) HEK293T cells were transfected with a plasmid expressing one of the ASFV-encoded pMGFs alone with 100 ng of a reporter plasmid and 5 ng of the pRL-TK plasmid for 24 h and then the cells were stimulated with poly(I:C) (1 μg). At 12 hpt, the cells were collected and the luciferase activities were measured. A p value of less than 0.05 was considered statistically significant. *p<0.05, **p<0.01, ***p<0.001.
Fig 4.
pMGF505-7R inhibits IL-1β and IFN-β production.
(A) HEK293T cells were transfected with increasing doses of a plasmid expressing pMGF505-7R in the presence of the iGLuc-based NLRP3 inflammasome system, and the supernatants were assessed for luciferase activity at 24 hpt. (B-F) HEK293T cells were co-transfected with increasing doses of plasmids expressing pMGF505-7R, along with NF-κB (B), IFN-α (C), IFN-β (D), ISRE (E), or ISG56 (F) promoter reporter, and then the cells were stimulated with cGAS/STING for 24 h, the cells were then collected, and the luciferase activities were measured. A p value of less than 0.05 was considered statistically significant. *p<0.05, **p<0.01, ***p<0.001.
Fig 5.
ASFV-Δ7R induces higher type I IFN and IL-1β production compared with ASFV-WT.
(A-D) PAMs were either mock-infected or infected with ASFV HLJ/18 (ASFV-WT) or ASFV-Δ7R at an MOI of 0.5. At 24 or 48 hpi, the IL-1β (A) and IFN-β (B) levels in the cell culture supernatants were detected by ELISA and the mRNA levels in the cell lysates were determined by qPCR. The genomic copy numbers of ASFV in PAMs were measured by qPCR (C) and the cell viabilities were analyzed using a CCK-8 counting kit (D). A p value of less than 0.05 was considered statistically significant. *p<0.05, **p<0.01, ***p<0.001.
Fig 6.
ASFV pMGF505-7R interacts with IKK complex to inhibit IL-1β transcription.
(A–C) HEK293T cells were transfected with NF-κB reporter, increasing amounts of a plasmid encoding pMGF505-7R along with stimulation of LPS (100 ng/mL) (A) or a plasmid encoding IKKα (B) or IKKβ (C). The luciferase activities were measured. The expressions of IKKα, IKKβ and pMGF505-7R were detected by Western blotting. GAPDH expressions were detected as a loading control. (D) HEK293T cells were transfected with a plasmid encoding Flag-tagged pMGF505-7R along with a plasmid encoding HA-tagged IKKα, IKKβ or NEMO as indicated. 36 hpt, the cells were lysed and whole cell lysates (WCL) were immunoprecipitated with anti-Flag mAb. The immunoprecipitants were detected by Western blotting with antibodies indicated. (E) PAMs were either mock-infected or infected with ASFV-EGFP-7R (1 MOI) for 36 h, and then a Co-IP was performed with anti-EGFP antibody. Immunoglobulin G (IgG) was used as a negative control. (F) PAMs were infected with ASFV-WT (1 MOI) or ASFV-Δ7R (1 MOI) for 12 or 24 h, and the phosphorylation levels of IκBα were analyzed by Western blot. The relative protein band intensities analyzed by Image studio. (G) PAMs were treated with TNF-α (15 ng/mL) or infected with ASFV-WT (WT) or ASFV-Δ7R (Δ7R), and p65 in the nuclear and cytoplasmic compartments, and ASFV-p30 were detected by Western blotting. Histone H3 and GAPDH were used as nuclear and cytosolic markers. A p value of less than 0.05 was considered statistically significant. *p<0.05, **p<0.01, ***p<0.001.
Fig 7.
pMGF505-7R targets NLRP3 to inhibit inflammasome complex formation.
(A) HEK293T cells were transfected with different doses of MGF505-7R in the presence of the iGLuc-based NLRP3 inflammasome system or the system lacking NLRP3 or both NLRP3 and ASC, and the supernatants were assessed for luciferase activity at 24 h after transfection. (B) HEK293T cells were transfected with a plasmid encoding pMGF505-7R along with a plasmid encoding HA-tagged NLRP3, ASC or caspase-1. 36 hpt, the cells were lysed and whole cell lysates (WCL) were immunoprecipitated with anti-Flag mAb. The immunoprecipitants were detected by immunoblotting with antibodies indicated. (C) PAMs were either mock-infected or infected with ASFV-EGFP-7R (1 MOI) for 36 h, and then a Co-IP was performed with anti-NLRP3 antibody. IgG was used as a negative control. (D) The PAM cell line 3D4/21 (CRL-2843) were transfected with a plasmid encoding GFP-ASC alone or together with Flag-MGF505-7R. At 24 hpt, cells were stimulated with Nigericin (5 μM) for another 4 h. The cells were then fixed and probed with rabbit anti-Flag mAb and GFP, and nucleus marker DAPI, and then observed by confocal microscopy. (E) HEK293T cells were transfected with GFP-ASC, HA-NLRP3 or Flag-MGF505-7R. 24 hpt, the cells were harvested, fixed, and the fraction of cells containing ASC specks was quantified by flow cytometry. Average values of cells with ASC specks came from three independent experiments. (F) PAMs were infected with ASFV-Δ7R or ASFV-WT. The cell lysates were prepared and the pellets were washed with PBS for three times and cross-linked using fresh DSS for Western blotting. A p value of less than 0.05 was considered statistically significant. *p<0.05, **p<0.01, ***p<0.001.
Fig 8.
pMGF505-7R targets IRF3 to inhibit IFN-β production.
(A-C) HEK293T cells were transfected with IFN-β reporter, different amounts (0, 100, 200 or 400 ng) of a plasmid encoding pMGF505-7R, together with a plasmid encoding TBK1 (A), IRF3 (B) or IRF3-5D (C). 24 hpt, the cells were lysed and the activities of the IFN-β reporter were assessed by the luciferase assay. The expressions of TBK1, IRF3 and pMGF505-7R were detected by Western blotting. GAPDH served as a loading control. (D-E) HEK293T cells were transfected with a plasmid encoding pMGF505-7R along with a plasmid encoding HA-IRF3 or HA-IRF3-5D as indicated. 36 hpt, the cells were lysed and whole cell lysates were immunoprecipitated with anti-Flag mAb. The immunoprecipitants were detected by immunoblotting with antibodies indicated. (F) PAMs were either mock-infected or infected with ASFV-EGFP-7R for 36 h, and then a Co-IP was performed with anti-IRF3 antibody. IgG was used as a negative control. (G) HeLa cells were transfected with GFP-IRF3 and Flag-MGF505-7R. At 24 hpt, the cells were stimulated with SeV for another 12 h and then probed with rabbit anti-Flag mAb and GFP, stained with nucleus marker DAPI and subsequently observed by confocal microscopy. The percentages of IRF3 nuclear translocation were quantified. (H) PAMs were infected with SeV, ASFV-WT (WT) or ASFV-Δ7R (Δ7R), and IRF3 in the nuclear and cytoplasmic compartments and ASFV-p30 were detected by Western blotting. Histone H3 and GAPDH were used as nuclear and cytosolic markers. A p value of less than 0.05 was considered statistically significant. *p<0.05, **p<0.01, ***p<0.001.
Fig 9.
Deletion of MGF505-7R attenuates ASFV virulence in pigs.
(A-C) The rectal temperature measurements (A) and survival rates (B) for the different groups of pigs unchallenged (Ctrl, n = 4), or challenged with 103 HAD50 of parental ASFV-WT (WT, n = 4), 103 HAD50 of ASFV-Δ7R (Δ7R, n = 7) or 105 HAD50 of ASFV-Δ7R (Δ7R, n = 7) were recorded daily till day 19 post challenge. At day 19, the surviving pigs were euthanized, and the blood and tissues including the heart, lung, spleen, tonsil, thymus and five lymph nodes (intestinal lymph node, inguinal lymph node, submaxillary lymph node, bronchial lymph node, and gastrohepatic lymph node) were collected from all pigs for viral DNA quantification by qPCR (C).
Fig 10.
ASFV-Δ7R infection induces higher type I IFN and IL-1β production in pigs.
(A-D) Pigs were mock challenged with PBS (Ctrl, n = 4) or challenged intramuscularly with ASFV-Δ7R (Δ7R, n = 5, 103 and 105 HAD50) or parental ASFV-WT (WT, n = 4, 103 HAD50), and the sera samples were collected at day 1, day 5 and day 8 post challenge. The protein levels of IFN-β (A), IFN-α (B) and IL-1β (C) in the serum samples were measured with commercial ELISA kits. The virus loads in the serum samples collected at day 1, day 5 and day 8 post challenge. were evaluated using qPCR. (D) Data presented are mean ± SD. **, P < 0.01, *, P < 0.05, student’s t-test, in comparison with control groups during statistical analysis.
Fig 11.
Schematic model of pMGF505-7R inhibits IL-1β and type I IFN production.
After ASFV infection, pMGF505-7R inhibits IL-1β and IFN-β production. Mechanistically, pMGF505-7R interacts with IKK complex to inhibit NF-κB activation and binds to NLRP3 to inhibit inflammasome formation, leading to decrease IL-1β production. Moreover, pMGF505-7R inhibits the nuclear translocation of IRF3 to block type I IFN production.