Fig 2: PRV VP22 inhibits ZBP1-mediated inflammatory responses. (A) THP-1 cells were transfected with 2 µg of a plasmid promoting the expression of MYC-tagged RIPK3 (MYC-RIPK3) and 2 µg of a plasmid promoting the expression of HA-ZBP1, together with 2 µg of either an empty vector, a plasmid driving either the expression of Flag-VP22, or that of Flag-GFP, and then stimulated with Lipopolysaccharide (LPS, 1 µg/mL) plus Nigericin (NG, 2 µM). At 24 hpt, cells were processed for IP with anti-HA magnetic beads. WCLs and precipitated proteins were analyzed by western blots with antibodies against MYC-tag, HA-tag, Flag-tag, and GAPDH. (B) BMDMs were mock infected or infected with PRV-WT, ΔVP22, or ΔVP22R at 5 PFU/cell. At 12 hpi, cells were processed for IP with anti-ZBP1 or anti-RIPK3. WCLs and precipitated proteins were analyzed by western blots with antibodies against ZBP1, RIPK3, Caspase-8, VP16, VP22, and GAPDH. (C) Zbp1+/+ and Zbp1−/− BMDM cells were mock infected or infected with PRV-WT, ΔVP22, or ΔVP22R at 5 PFU/cell. At 12 hpi, cells were harvested for western blot analysis with antibodies against ZBP1, VP22, NLRP3, ASC, Caspase-1, VP16, and GAPDH. The intensities of the western blot bands were analyzed using ImageJ. The vector or mock group was set as 1.00. All the data are representative of at least three independent experiments with similar results.

Fig 3: The N-terminal 1–50 aa domain of PRV VP22 is responsible for inhibiting ZBP1-mediated inflammatory responses. (A) Schematic representation of WT and truncated recombinant ZBP1 proteins. HEK-293 T cells were transfected with 2 µg of either Flag-empty vector, Flag-VP22, or one of its truncated mutants [Flag-VP22 (50–246 aa), Flag-VP22 (100–246 aa), or Flag-VP22 (1–200 aa)]. At 24 hpt, cells were harvested for western blot analysis with antibodies against Flag-tag and GAPDH. (B) HEK-293 T cells were transfected with 2 µg of a vector promoting the expression of HA-ZBP1 together with 2 µg of either a Flag-empty vector or a plasmid encoding Flag-VP22 (full length), Flag-VP22 (50–246 aa), Flag-VP22 (100–246 aa), or Flag-VP22 (1–200 aa). At 24 hpt, cells were processed for IP with anti-Flag magnetic beads. WCLs and precipitated proteins were analyzed by western blots with antibodies against Flag-tag, HA-tag, and GAPDH. (C) THP-1 cells were transfected with 2 µg of a plasmid encoding MYC-RIPK3 and 2 µg of a plasmid encoding HA-ZBP1, together with 2 µg of either a Flag-empty vector or a plasmid encoding Flag-VP22 (full length), Flag-VP22 (50–246 aa), Flag-VP22 (100–246 aa), or Flag-VP22 (1–200 aa) and then stimulated with LPS (1 µg/mL) plus NG (2 µM). At 24 hpt, cells were processed for IP with anti-HA magnetic beads. WCLs and precipitated proteins were analyzed by western blot with antibodies against MYC-tag, HA-tag, Flag-tag, and GAPDH. (D) BMDMs were mock infected or infected with PRV-WT, ΔVP22, or ΔVP22R at 5 PFU/cell. At 12 hpi, cells were processed for IP with anti-ZBP1 or anti-RIPK3. WCLs and precipitated proteins were analyzed by western blots with antibodies against ZBP1, RIPK3, Caspase-8, VP16, and GAPDH. (E) Zbp1+/+ and Zbp1−/− BMDMs were mock infected or infected with PRV-WT, ΔVP22, ΔVP22 (1–50 aa), ΔVP22 (51–246 aa), or ΔVP22R at 5 PFU/cell. At 12 hpi, cells were harvested for western blot analysis with antibodies against ZBP1, NLRP3, ASC, Caspase-1, VP16, and GAPDH. The intensities of the western blot bands were analyzed using ImageJ. The vector or mock group was set as 1.00. All the data are representative of at least three independent experiments with similar results.

Fig 4: ZBP1 inhibits PRV viral replication and pathogenicity. (A and B) Effects of ZBP1 on PRV viral replication. (A) WT SH-SY5Y, 3D4/2, N2a cells and corresponding Zbp1-knockdown cells or (B) Zbp1+/+ (WT) and Zbp1−/− MEFs were infected with PRV-WT, ΔVP22, or ΔVP22R at 0.01 MOI. At 48 hpi, cells and supernatants were harvested, and total virus titers were determined by plaque assay in Vero cells. (C) Comparison of the survival curves obtained for Zbp1+/+ (WT) and Zbp1−/− mice, which were either mock infected or infected with 1 × 105 PFU/mice of PRV-WT, ΔVP22, or ΔVP22R by subcutaneous injection (eight mice/group). Simple survival analyses for the four groups using Kaplan–Meier curves (Zbp1+/+: P < 0.0001; Zbp1−/−: P = 0.011). (D) Four groups of mice (four mice/group) treated as in C were sacrificed at 3 dpi and assessed by RT-PCR for viral DNA load in brain and lung. (E) Brain and lung sections from mice infected with different PRVs (1 × 105 PFU/mice) were analyzed by H&E staining at 3 dpi. The magnified area of each section (right-hand side) is localized by a square on the corresponding lower magnification picture (left-hand side) (scale bar: 100 µm). Data are representative of at least three independent experiments with similar results (mean ± SD of n = 3 biological replicates in A and B, n = 4 biological replicates in D). *P < 0.05 and **P < 0.01 (two-tailed unpaired t test).

Fig 5: RNA-seq analysis revealed genes differentially expressed during infection with PRV-WT and ΔVP22 in WT or Zbp1-deficient BMDMs. (A) GSEA hallmark analysis of the upregulated or downregulated genes. (B) Heatmap showing the relative expression levels of genes of the inflammatory pathway in mock-infected and PRV-WT or ΔVP22-infected Zbp1+/+ and Zbp1−/− BMDMs. (C) Zbp1+/+ and Zbp1−/− BMDMs were infected with PRV-WT, ΔVP22, or ΔVP22R at an MOI of 5; at 12 hpi, the cells were harvested for RNA extraction and RT-PCR quantification of IL-1β and Nlrp3 mRNAs and 18s rRNA. Data are representative of at least three independent experiments with similar results (mean ± SD of n = 3 biological replicates). **P < 0.01 (two-tailed unpaired t test).