Fig 1.
PRV UL4 is a promotor of NLRP3 inflammasome Activation.
(A) ELISA was used to detect the effect of viral protein on IL-1β secretion in 293T cells reconstructed with NLRP3 inflammasome system. 293T cells were co-transfected with NLRP3 inflammasome system-associated plasmids encoding porcine NLRP3, ASC, procaspase-1, Pro-IL-1β, along with the plasmids encoding the indicated viral protein or empty vector (pEGFP-N1) plasmids for 24 h, then the cells were treated with LPS (1mg/mL) for 1 h and further stimulated with nigericin (2 μM) for another 6 h. (B, C) The role of UL4 on NLRP3 inflammasome system reconstructed in 293T cells. 293T cells were co-transfected with NLRP3 inflammasome system-associated plasmids, along with plasmids expressing different concentrations of GFP-UL4, or pEGFP-N1 (Vec) or without transfection (NT) for 24 h. Then, the cells were stimulated without (NS) or with LPS/NG. (D-I) The role of UL4 on NLRP3 inflammasome in 3D4/21 cells or BMDMs. 3D4/21 cells (D-F) or BMDMs (H-G) were transfected with plasmids encoding GFP-UL4 or Vec for 24 h, the cells were stimulated without (NS) or with LPS/NG or LPS/ATP. Precipitated conditioned media (Sup) and whole cell lysates (WCL) were analyzed by immunoblotting to detect expression levels of the indicated proteins (B, D, G), and the cell-free supernatants were subjected to ELISA for the secretion of IL-1β (C, E, H) and IL-18 (F, I). * P < 0.05; ** P < 0.01, compared with the Vec transfected cells with the same treatment.
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.
The UL4 null mutation alleviates inflammasome activation in PRV-infected cells.
(A, B) Construction and identification of PRV-ΔUL4. The construction flowchart of UL4 gene deletion virus strain (A). 3D4/21 cells were infected with Mock, PRV-WT, or PRV-ΔUL4 (5 MOI) for 12 h, then the expression of UL4 and GFP was detected by immunoblotting (B). (C-H) The effects of UL4 null mutation on the secretion of IL-1β and IL-18 in PRV-infected cells. 3D4/21 cells (C, D) or BMDMs (E, F) were infected with Mock, PRV-WT, or PRV-ΔUL4 (5 MOI) for 12 h. or treated with LPS/NG as the positive control. ELISA assay for IL-1β (C, E) and IL-18 (D, F) in supernatants was measured, and Sup and WCL were analyzed by immunoblotting for indicated protein (G, H). ** P < 0.01, compared with the Mock-infected cells (C-F); ## P < 0.01, versus the PRV-WT-infected cells (C-F).
Fig 4.
Viral UL4 protein promotes pyroptosis in macrophage cells.
(A-D) Effect of UL4 on cell pyroptosis. 3D4/21 (A, B) or BMDMs (C, D) were transfected with plasmids encoding GFP-UL4 or GFP for 24 h, then the 3D4/21 cells were treated without (NS) or with LPS/NG; the BMDMs were treated without (NS) or with the Poly dA:dT. (E-H) Effect of UL4 deletion on cell pyroptosis. 3D4/21 cells (E, F) or BMDMs (G, H) were infected with Mock, PRV-WT, PRV-ΔUL4 (5 MOI) for 12 h. (I-L) Effect of UL4 repair on cell pyroptosis. 3D4/21 cells (I, J) or BMDMs (K, L) were infected with Mock, PRV-WT, and PRV-UL4repair (5 MOI) for 12 h. Supernatants were collected to analyze the LDH activity (A, C, E, G, I, K); cell lysates were analyzed by immunoblotting to detect the indicated proteins (B, D, F, H, J, L). ** P < 0.01, compared with the Vec transfected cells (A, C) or the Mock-infected cells (E, G, I, K). ## P < 0.01, compared with the PRV-WT infected cells with the same treatment (E, G, I, K).
Fig 5.
(A-C) Interaction between the PRV UL4 and ASC was detected by Co-IP. 293T cells were co-transfected with plasmids expressing Flag-ctrl, Flag-NLRP3, Flag-CASP1, or Flag-ASC, along with GFP-UL4 for 24 h. Whole-cell lysates (WCL) were immunoprecipitated using the indicated antibody, followed by immunoblotting with indicated antibodies (A). 293T cells were transiently transfected with the indicated plasmids for 24 h and the interaction between GFP-UL4 and Flag-ASC was verified by Co-IP analysis (B). 3D4/21 cells were infected with Mock or PRV (5 MOI) for 12 h. WCL were immunoprecipitated with an anti-ASC antibody, followed by immunoblotting using indicated antibodies (C). (D) IFA analysis of the interaction between GFP-UL4 and Flag-NLRP3, Flag-ASC, and Flag-CASP1 in 293T cells co-transfected with indicated plasmids for 24 h, then visualized with confocal microscopy. Scale bar, 10 μm.
Fig 6.
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 7.
The UL4 mutation alleviates inflammasome activation and pyroptosis in PRV-infected cells.
(A, B) Construction and identification of PRV-UL4mut. The construction flowchart of UL4 gene mutation virus strain (A). 3D4/21 cells were infected with Mock, PRV-WT, or PRV-UL4mut (5 MOI) for 12 h, then the expression of UL4 and GFP was detected by immunoblotting (B). (C-H) The effects of UL4 mutation on the secretion of IL-1β and IL-18 in PRV-infected cells. 3D4/21 cells (C, D, G) or BMDMs (E, F, H) were infected with Mock, PRV-WT, or PRV-UL4mut (5 MOI) for 12 h. (I-L) Effect of UL4 mutation on cell pyroptosis. 3D4/21 cells (I, K) or BMDMs (J, L) were infected with Mock, PRV-WT, PRV-UL4mut (5 MOI) for 12 h. ELISA assay for IL-1β (C, E) and IL-18 (D, F) in supernatants was measured, and Sup and WCL were analyzed by immunoblotting for indicated protein (G, H). Supernatants were collected to analyze the LDH activity (I, J); cell lysates were analyzed by immunoblotting to detect the indicated proteins (K, L). ** P < 0.01, compared with the Mock-infected cells, ## P < 0.01, versus the PRV-WT-infected cells.
Fig 8.
PRV UL4 augments ASC oligomerization and speck formation.
(A, B) Detection of the effect of UL4 on NLRP3 inflammasome. 3D4/21 cells co-transfected with pEGFP-N1 or pEGFP-UL4 for 24 h were lysed, and the indicated protein expression levels were analyzed by immunoblotting (A). 3D4/21 cells were co-transfected with different concentrations of pEGFP-UL4 for 24 h, followed by stimulation of LPS/NG (B). WCL were immunoprecipitated using anti-ASC antibodies and analyzed using the indicated antibody. (C, D) The effect of UL4 on the formation of ASC speck. 293T cells were co-transfected plasmids encoding Flag-ASC with Vec (PCMV-HA) or plasmids encoding HA-UL4 for 24 h, ASC specks were detected by immunofluorescence staining using anti-Flag. Bars, 50 μm (C); Percentage of cells with ASC speck structures in the experiments in C (D). Quantified data are presented as mean ± SEM of the percentage of cells with ASC speck formation based on biological replicates. (E-H) The effect of UL4 on ASC oligomerization. 3D4/21 cells (E) or BMDMs (F) were respectively transfected with plasmids expressing UL4mut, UL4, or GFP (Vec), followed by stimulation of LPS/NG (E) or Poly dA:dT (F); 3D4/ 21 cells (G) or BMDMs (H) were infected with Mock, PRV-WT or PRV-UL4mut for 12 h; then ASC in disuccinimidyl suberate (DSS)–treated Triton-insoluble fractions of indicated cells was analyzed by immunoblotting. ** P < 0.01, compared with the Vec transfected cells.
Fig 9.
PRV UL4 protein facilitates ASC oligomerization and speck formation by promoting ASC phosphorylation.
(A-C) The effect of PRV UL4 on the polyubiquitination level of ASC. Immunoprecipitation analysis of ASC ubiquitination in 293T cells transfected with plasmids encoding Flag-ASC, GFP-UL4, HA-tagged WT, or mutant ubiquitin harboring lysine-to-arginine mutations in all lysine residues except lysine 63 (ubiquitin K63) for 24 h (A); Immunoprecipitation analysis of ASC ubiquitination in 3D4/21 cells (B) and BMDMs (C) infected with Mock, PRV-WT, PRV-UL4mut for 12 h. (D-G) The effect of PRV UL4 on the phosphorylation level of ASC Y144 (equivalently l to porcine Y146). Immunoprecipitation analysis of ASC Y146 phosphorylation in 3D4/21 cells or ASC Y144 phosphorylation in BMDMs, transfected with plasmids encoding UL4mut, UL4, or Vec for 24 h, followed by stimulation of LPS/NG (D) or Poly dA:dT (E); Immunoprecipitation analysis of ASC Y146 phosphorylation in 3D4/21 cells (F) or ASC Y144 phosphorylation in BMDMs (G), infected with Mock, PRV-WT, PRV-UL4mut for 12 h. (H-K) The effect of PRV UL4 on the phosphorylation level of JNK and SYK. Western blotting analysis of indicated proteins in 3D4/21 cells (H) or BMDMs (I) were transfected with plasmids encoding UL4mut, UL4, or GFP for 24 h, followed by stimulation of LPS/NG (H) or Poly dA:dT (I). Western blotting analysis of indicated proteins in 3D4/21 cells (J) or BMDMs (K) infected with Mock, PRV-WT, PRV-UL4mut for 12 h.
Fig 10.
The UL4 mutation alleviates inflammation in PRV-infected pigs.
pigs (3 weeks old) were nasal infections with Mock, PRV-WT, or PRV-UL4mut at 108 TCID50/pig. (A) clinical score of pigs infected with Mock, PRV, or PRV-UL4mut at postinfection 48 h (n = 4 per group). (B-E) The effects of UL4 mutation on the secretion of IL-1β (B, C) or the IL-18 (D, E) in the peripheral blood (B, C) or the lung and brain tissues of mice (D, E) was detected by ELISA at postinfection 24 h. (F) The effects of UL4 mutation on the GSDMD-NT in lung and brain tissues were detected by western blotting at postinfection 24 h. (G, H) PFU in the indicated organs on indicated times. (I) Histopathology analysis (H&E staining) of pigs from the lungs and brain tissues was shown. ** P < 0.01, versus the Mock-infected group (B, C, D, E); ## P < 0.01, compared with the PRV-WT infected group with the same treatment (A, B, C, D, E, G, H).
Table 1.
Comparison of histological lesion scores of lungs and brains from PRV-WT and PRV-UL4mut infected piglets.
Table 2.
Comparison of histological lesion scores of lungs and brains from PRV-WT and PRV-UL4mut infected mice.