Coronavirus Nsp5‑mediated dual‑site cleavage of GSDMA modifies its antiviral and proinflammatory functions

Figures

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  Fig 1. CoV nsp5 cleaves GSDMA at residues Q187 and Q247. (A) IPI-2I cells were transfected with eukaryotic expression plasmids encoding HA-tagged PDCoV nsp5_WT or PDCoV nsp5_C144A. Cells were lysed at 48 h post-transfection (hpt), and the expression levels of endogenous full-length porcine GSDMA (pGSDMA_FL) and cleaved pGSDMA were determined by immunoblotting using an anti-pGSDMA antibody. β-Tubulin was used as a loading control. (B) Recombinant SUMO-tagged pGSDMA protein (pGSDMA_FL-SUMO; 12.5 μg) was incubated with purified PDCoV nsp5_WT or PDCoV nsp5_C144A proteins (1 μg each) for 30 min. The levels of pGSDMA_FL-SUMO and cleaved pGSDMA-SUMO were analyzed by immunoblotting using an anti-pGSDMA antibody. (C) Amino acid sequence logos of the PDCoV nsp5 auto-cleavage sequences (nsp5-nsp16) were created using PSSMSearch, and the height of the letters represents the relative frequency of the amino acids. (D) Cells were co-transfected with eukaryotic expression plasmids encoding HA-tagged PDCoV nsp5 and Myc-tagged pGSDMA_FL (wild-type pGSDMA_FL, abbreviated as pGSDMA_WT) or pGSDMA_FL mutants (pGSDMA_Q187A, pGSDMA_Q247A, and pGSDMA_Q187A&Q247A). (E) Cells were co-transfected with eukaryotic…

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  Fig 2. CoV infections cause the cleavage of GSDMA both in vivo and in vitro. (A and C) IPI-2I cells were infected with PDCoV (0.05, 0.1, and 0.5 MOI) (A) or with PEDV (0.25 MOI) (C), and lysed for immunoblotting at 24 hours post-infection (hpi). (B and D) Small intestinal tissues from piglets, either mock-infected or infected with PDCoV (B) or PEDV (D), were harvested and lysed for immunoblotting. (E) iBMDMs were infected with MHV (0.5 MOI) and lysed for immunoblotting at 8 hpi. (F) Liver tissues from mice, either mock-infected or infected with MHV, were harvested and lysed for immunoblotting. (A-F) Endogenous GSDMA_FL and cleaved GSDMA were assessed using anti-GSDMA antibodies. β-Tubulin was used as a loading control.

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  Fig 3. GSDMA_1-247 contributes to GSDMA-mediated inflammatory responses. (A) Schematic representation of GSDMA and its cleavage fragments induced by CoV nsp5. (B, D, F, and G) IPI-2I cells were transfected with eukaryotic plasmids encoding pGSDMA_1-247, pGSDMA_1-187, and pGSDMA_FL. (B and D) Cell supernatants and/or cell lysates were then collected at 48 hpt. (F and G) Cells were then infected with PDCoV (0.5 MOI) (F) or PEDV (0.25 MOI) (G) for an additional 24 h, after which the cell supernatants were collected. (B) LDH release and IL-1β levels in the cell supernatants, as well as intracellular ATP cell viability, were analyzed using assay kits. (D, F, and G) The protein levels of porcine proinflammatory cytokines (TNF-α, IL-6, and IL-8) in the cell supernatants were assessed through ELISA. (C, E, and H) iBMDMs were transfected with eukaryotic plasmids encoding mGSDMA_1-247, mGSDMA_1-187, or mGSDMA_FL. (C, E) Cell supernatants and/or cell lysates were collected at 48 hpt. (H) Cells were subsequently infected with MHV (0.5 MOI) for an additional 8 h, and the cell supernatants were then collected. (C) LDH release and IL-1β levels in the cell supernatants, as well as intracellular ATP…

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  Fig 4. GSDMA_1-247 is responsible for the antiviral activity of GSDMA. (A-C) IPI-2I cells (A and B) or iBMDMs (C) were transfected with GSDMA-specific siRNA (siGSDMA) or control siRNA (siNC) for 36 h, followed by infection with PDCoV (0.5 MOI) for an additional 24 h (A), PEDV (0.25 MOI) for an additional 24 h (B), or MHV (0.5 MOI) for an additional 8 h (C). (D-F) IPI-2I cells (D and E) or iBMDMs (F) were transfected with eukaryotic expression plasmids encoding GSDMA_1-247, GSDMA_1-187, or GSDMA_FL. At 48 hpt, cells were subsequently infected with PDCoV (0.5 MOI) for an additional 24 h (D), PEDV (0.25 MOI) for an additional 24 h (E), or MHV (0.5 MOI) for an additional 8 h (F). (A-F) Total RNA extracted from cell lysates was analyzed by RT‑qPCR, and viral titers in cell supernatants were determined by the TCID₅₀ assay. Data analysis was performed using Student’s t-test or one-way ANOVA. Viral RNA copy number was measured using RT-qPCR. Values are shown as the mean ± SD from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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  Fig 5. GSDMA knockout reduces inflammation and mortality in vivo. (A) Schematic representation of the animal experiment protocol. Groups of six WT or Gsdma-/- mice were inoculated with 1 × 106 PFU of MHV via intraperitoneal injection. The mortality (B) and body weight changes (C) of the mice were recorded over a consecutive ten-day period. (D) Schematic representation of the animal experiment protocol. Groups of six WT or Gsdma-/- mice were inoculated with either DMEM as a negative control or 1 × 106 PFU of MHV via intraperitoneal injection. Three mice from each group were euthanized at 3 and 5 dpi, respectively. Liver tissues were collected for virus titration, pathological analysis, and ELISA, while blood samples were collected for ELISA. (E-G) Liver tissues from mice euthanized at 5 dpi were analyzed for viral RNA copy number and titers in L929 cells (E), gross lesions (F), and pathological changes (G). (H and I) Serum and liver tissues from mice euthanized at 5 dpi were collected to assess TNF-α (H) and IL-6 (I) protein levels via ELISA. (E, H, and I) Data analysis was performed using Student’s t-test (E) or one-way ANOVA (H and I). Values are shown as the mean ± SD. *, p < 0.05;…