Fig 2: IRF1 is dispensable for cell death in response to classical inflammasome-inducing ligands.A–C, representative images of cell death and IL-18 release in WT, Irf1−/−, and Nlrp3−/− bone marrow–derived macrophages (BMDMs) at 1 h after stimulation with LPS plus ATP (A and C) and real-time analysis of cell death (B). D–F, representative images of cell death and IL-18 release in WT, Irf1−/−, and Casp11−/− BMDMs at 6 h post-transfection with LPS (D and F) and real-time analysis of cell death (E). G–I, representative images of cell death and IL-18 release in WT, Irf1−/−, and Aim2−/− BMDMs at 4 h post-transfection with poly(dA:dT) (G and I) and real-time analysis of cell death (H). J–L, representative images of cell death and IL-18 release in WT, Irf1−/−, and Nlrc4−/− BMDMs at 6 h post-transfection with flagellin (J and L) and real-time analysis of cell death (K). Data are representative of at least three independent biological repeats (A and B; D and E; G and H; J and K), and individual datapoints from independent biological replicates are shown (C, F, I, and L). Scale bar is representative of 50 μm. IL-18, interleukin 18; IRF1, IFN regulatory factor 1; LPS, lipopolysaccharide; ns, not significant.

Fig 3: Sequence of UL4 is involved in inflammasome activation.(A-C) Identification of the truncated UL4 expression. Schematic diagram of PRV UL4 protein and truncated mutants UL4 protein (A). Western blotting detected the expression of UL4 and truncated mutants UL4 using GFP antibody (B, C). (D, E) Interaction between the truncated UL4 and ASC was detected by IFA and Co-IP. IFA analysis of the interaction between truncated mutants UL4 and Flag-ASC in 293T cells co-transfected with indicated plasmids for 24 h, then visualized with confocal microscopy (D). 293T cells were co-transfected with plasmids expressing porcine ASC and plasmids expressing UL4 protein or truncated UL4 protein as indicated for 24 h (E). WCL were immunoprecipitated with the indicated antibody, followed by immunoblotting using indicated antibodies. (F-I) Identification of key domains of UL4 and ASC interaction. Alignment diagram of Herpesvirus UL4 amino acid partial sequence (F). Schematic diagram of UL4 mutant (G). IFA analysis of the interaction between GFP-UL4mut and Flag-ASC in 293T cells co-transfected with indicated plasmids for 24 h, then visualized with confocal microscopy (H). 293T cells were co-transfected with the indicated plasmids for 24 h. WCL were immunoprecipitated with the indicated antibody, followed by immunoblotting using indicated antibodies (I). (J-M) Identification of UL4 key domains that promote the activation of NLRP3 inflammasome and AIM2 inflammasome. 3D4/21 cells were respectively transfected with plasmids expressing UL4 protein or truncated mutants UL4 protein for 24 h, followed by the stimulation of LPS/NG (J, K). BMDMs were respectively transfected with plasmids expressing UL4 protein or truncated mutants UL4 protein for 24 h, followed by the stimulation of Poly dA:dT (L, M). Supernatants were subjected to ELISA for IL-1β secretion (J, L). Sup and WCL were analyzed by immunoblotting to detect the indicated proteins (K, M). ** P < 0.01, compared with the cells expressing UL4. Scale bar, 10 μm.

Fig 4: PRV UL4 also promotes AIM2-inflammasome activation.(A, B) The role of UL4 on the activation of AIM2 inflammasome reconstructed in 293T cells. 293T cells were transfected with AIM2 inflammasome system-associated plasmids expressing murine AIM2, ASC, pro-CASP1, Pro-IL-1β, along with plasmids encoding GFP-UL4 or GFP (Vec) for 24 h, then transfected without (NS) or with or Poly dA:dT (2 μM). (C-E) The role of UL4 on the activation of AIM2 inflammasome in BMDMs. BMDMs were transfected with plasmids expressing GFP-UL4 or GFP (Vec) for 24 h, then transfected without (NS) or with Poly dA:dT. (F-J) The effect of the different viral UL4 on inflammasome. The expression of the indicated viral UL4 protein was measured by western blotting (F). The effect of the different viral UL4 on NLRP3 inflammasome (G, I) or AIM2 inflammasome (H, J). 3D4/21 cells (G, I) and BMDMs (H, J) transfected with plasmids expressing the indicated viral UL4 protein for 24 h, followed by the stimulation of LPS/NG (G, I) or Poly dA:dT (H, I). Supernatants were subjected to ELISA for IL-1β secretion (A, D, G, H) or IL-18 secretion (E). Sup and WCL were analyzed by immunoblotting to detect the indicated proteins (B, C, I, J). ** P < 0.01, compared with the empty vector-expressed cells with the same treatment (A, D, E, G, H); # P < 0.05, ## P < 0.01, versus the PRV-WT-infected cells (G, H).