The Very Virulent IBDV Viral Protein VP3 Promotes the Caspase-3 Mediated Cleavage of GSDME

Figures

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  Figure 1. Detection of tissue distribution of GSDME in SPF chickens infected with vvIBDV. (A) The viral copy number in the bursa of Fabricius at different time points after IBDV infection (3 days, 4 days, and 5 days). The viral load in the bursa of Fabricius was detected by an absolute fluorescence quantitative method at different infection times, and reached the peak on the 5th day. (B) The relative levels of viral copy number in different organs and tissues after infection. (C,D) The relative expression levels of GSDME mRNA in different immune organs and tissues after infection. The transcription levels of GSDME and IL-1β in each organ on the 5th day after IBDV infection were detected. The expression was significantly upregulated in the bursa of Fabricius and kidney, while there was no significant change in the thymus, liver, and spleen. (E) Gx infection induces the activation of GSDME in DT40 cells. Proteolytic cleavage of GSDME in Gx-infected DT40 cells was determined by Western blotting at 24 h post-infection (hpi). The abundance of Gx VP2 protein and β-actin as an internal control was also determined by Western blotting. The figures show the mean ± standard deviation, n = 3, * p <…

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  Figure 2. The IBDV viral protein cannot directly cleave GSDME. (A) Co-immunoprecipitation (Co-IP) analysis of the interaction between GSDME and IBDV viral proteins (VP1, VP2, VP3, VP4, VP5). HEK-293T cells were co-transfected with Flag-tagged GSDME and HA-tagged IBDV individual viral proteins. Cell lysates were immunoprecipitated with anti-Flag antibody, and the precipitated proteins were detected by Western blotting using anti-HA antibody. The results show that GSDME interacts with VP2, VP3, and VP4, but not with VP1 or VP5. (Original image is in Supplementary Materials Figure S2) (B) Confocal assays were used to assess the colocalization between GSDME and VP3 and VP4. HEK-293T cells were co-transfected with pFlag-GSDME and pHA-VP3 or pHA-VP4 for 36 h. Cells were incubated with anti-Flag mAb produced in mice and anti-HA mAb produced in rabbits; the interaction between GSDME and VP3 or VP4 was determined by confocal analysis. (C) The IBDV viral protein cannot directly induce pyroptosis through GSDME. HEK-293T cells were co-transfected with pFlag-GSDME and eukaryotic plasmids expressing viral protein-HA fusions containing VP2, VP3, and VP4 of vvIBDV. Twenty-four hours post-transfection,…

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  Figure 3. Identification of the specific Caspase-3 cleavage site of GSDME. (A) Schematic diagram showing aspartic acid residues in the middle domain of GSDME. (B) Detection of Caspase-3 cleavage ability after GSDME aspartic acid site mutation. pGSDMED270A, pGSDMED273A, and pGSDMED283A and co-transfected them with Caspase-3 into HEK-293T cells to detect the GSDME-N fragment. (Original image is in Supplementary Materials Figure S3) (C) Schematic diagram of truncated expression pattern before and after the GSDMED270 mutation site. (D) Verify the expression of the N-terminal and C-terminal regions of GSDME. (Original image is in Supplementary Materials Figure S3) (E) LDH release from the cells transfected with pMyc-Caspase-3, pFlag-GSDME and pHA-VP3 was measured as described in Materials and Methods. (F) After transfection of cells with pGSDME, pGSDME-N, and pGSDME-C, PI staining was performed. HEK-293T cells were transfected with each plasmid separately for 24 h, stained with PI for 5 min at room temperature, and observed under a fluorescence microscope. Red arrows indicate pyroptotic cells. The scale bar is 125 μm. (G) The proportion of PI-positive cells was detected using flow cytometry.…

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  Figure 4. VP3 promotes Caspase-3-mediated cleavage of GSDME. (A) Effects of IBDV proteins on Caspase-3-mediated GSDME cleavage were detected by Western blot. HEK-293T cells were co-transfected with pMyc-Caspase-3, pFlag-GSDME, and eukaryotic plasmids expressing viral protein-HA fusions containing VP1, VP2, VP3, VP4, or VP5 of IBDV. Twenty-four hours post-transfection, cell lysates were examined by Western blotting using anti-Myc, anti-HA, anti-Flag, and anti-β-actin antibodies; endogenous β-actin expression was used as an internal control. (Original image is in Supplementary Materials Figure S4) (B) PI staining showing pyroptotic cells in cultures expressing Caspase-3, GSDME, and VP3. After transfection of cells with pFlag-GSDME, pMyc-Caspase-3 and pHA-VP3 of vvIBDV. PI staining was performed. After separately transfecting each of the three plasmids into HEK-293T cells for 24 h, PI staining was carried out at room temperature for 5 min, and then observed under a fluorescence microscope. The scale bar is 75 μm. (C) Flow cytometry analysis after PI staining. (D) Detection of cleavage of GSDME and GSDMED270A. HEK-293T cells were co-transfected with pMyc-Caspase-3, pFlag-GSDME or…