pMGF505-7R determines pathogenicity of African swine fever virus infection by inhibiting IL-1β and type I IFN production
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.