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