Fig 2: 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).

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: IRF1 promotes AIM2- and ZBP1-dependent PANoptosis.A, representative images of cell death in WT, Irf1−/−, and Aim2−/− bone marrow–derived macrophages (BMDMs) at 8 h post-infection with HSV1 (10 MOI). B, real-time analysis of cell death in WT, Irf1−/−, and Aim2−/− BMDMs post-infection with HSV1 (10 MOI). C, immunoblot analysis of pro- (P45) and activated (P20) caspase-1 (CASP1); pro- (P54) and activated (P30) gasdermin D (GSDMD); pro- (P55) and cleaved (P44 and P18) caspase-8 (CASP8); pro- (P35) and cleaved (P20) caspase-7 (CASP7); pro- (P35) and cleaved (P19 and P17) caspase-3 (CASP3); phosphorylated mixed lineage kinase domain-like (pMLKL) and total MLKL (tMLKL) in HSV1–infected BMDMs at 8 h post-infection or BMDMs in media (Med). Immunoblot analysis of HMGB1 and LDH from the supernatant (Sup) of HSV1-infected BMDMs at 8 h post-infection or BMDMs in Med. D, representative images of cell death in WT, Irf1−/−, and Zbp1−/− BMDMs following stimulation with IFNγ plus KPT-330 at 20 h poststimulation. E, real-time analysis of cell death in WT, Irf1−/−, and Zbp1−/− BMDMs following stimulation with IFNγ plus KPT-330. F, immunoblot analysis of pro- and activated CASP1, pro- and activated GSDMD, pro- and cleaved CASP8, pro- and cleaved CASP7, pro- and cleaved CASP3, and pMLKL and tMLKL in IFNγ plus KPT-330–stimulated cells at 20 h poststimulation or BMDMs in Med. Immunoblot analysis of HMGB1 and LDH from the Sup of IFNγ plus KPT-330–stimulated cells at 20 h poststimulation or BMDMs in Med. Data are representative of at least three independent biological replicates. The dotted lines in immunoblot panels (C and F) are incorporated to visually separate media (Med) and treated conditions. Each antibody was probed on an individual blot, and samples from the same experiment were loaded to multiple gels (C and F). GAPDH was used as a single loading control for each set of samples. Analysis was performed using the two-way ANOVA (B and E). Scale bar is representative of 50 μm. AIM2, absent in melanoma-2; HMGB1, high mobility group box 1; HSV1, herpes simplex virus type 1; IFN, interferon; IRF1, IFN regulatory factor 1; LDH, lactate dehydrogenase; MOI, multiplicity of infection; ZBP1, Z-DNA binding protein 1.