Journal: Virologica Sinica

Article Title: The SARS-CoV-2 3CL protease inhibits pyroptosis through the cleavage of gasdermin D

doi: 10.1016/j.virs.2025.03.006

Figure Lengend Snippet: The SARS-CoV-2 3CL protease cleaves GSDMD at Q29 and Q193. A The top ten GSDMD cleavage sites predicted by NetCorona. B Comparison of known 3CL protease cleavage sites in Nsp4, Nsp6, and Nsp5 with predicted sites in GSDMD, the cleavage sites recognized by 3CL protease on different proteins were labeled in red. C The schematic illustrates the mutations of GSDMD Q29A, Q193A, and Q29A/Q193A, where the QS and QG motifs on the GSDMD protein, as predicted by NetConora, have been mutated to AS and AG, respectively, and these mutated sites are highlighted in red. D Western blot analysis of GSDMD cleavage by increasing amounts of GFP-3CL in 293T cells. E Western blot analysis was conducted to assess the cleavage of GSDMD in Huh7-ACE2 cells infected with SARS-CoV-2 at various multiplicities of infection (MOIs) of 0.01, 0.1, and 1. The exposure times for the GSDMD protein are indicated as short exposure for 1 ​s and long exposure for 5 ​s in this image. F Western blot analysis of wild-type and single mutant (Q29A or Q193A) GSDMD cleavage by GFP-3CL. G Western blot analysis of wild-type and double mutant (Q29A/Q193A) GSDMD cleavage by GFP-3CL. H and I Western blot analysis of Flag tagged GSDMB ( H ) and GSDME ( I ) cleavage by GFP-3CL of SARS-CoV-2 in 293T cells.

Article Snippet: The SARS-CoV-2 3CL protease inhibitor PF-00835231 (HY-137048) was purchased from MedChemExpress ( ).

Techniques: Comparison, Labeling, Western Blot, Infection, Mutagenesis

Journal: Virologica Sinica

Article Title: The SARS-CoV-2 3CL protease inhibits pyroptosis through the cleavage of gasdermin D

doi: 10.1016/j.virs.2025.03.006

Figure Lengend Snippet: 3CL protease activity is required for GSDMD cleavage. A Western blot analysis of GSDMD cleavage by GFP-3CL with or without 10 ​μM PF-00835231 treatment. B Dose-dependent inhibition of GSDMD cleavage by a series of 10-fold diluted PF-00835231 at concentrations of 1 ​μM, 10 ​μM and 100 ​μM. C Schematic of the 3CL protease C145A mutation. D Western blot analysis of GSDMD cleavage by the wild-type and C145A mutant 3CL proteases from SARS-CoV-2.

Article Snippet: The SARS-CoV-2 3CL protease inhibitor PF-00835231 (HY-137048) was purchased from MedChemExpress ( ).

Techniques: Activity Assay, Western Blot, Inhibition, Mutagenesis

Journal: Virologica Sinica

Article Title: The SARS-CoV-2 3CL protease inhibits pyroptosis through the cleavage of gasdermin D

doi: 10.1016/j.virs.2025.03.006

Figure Lengend Snippet: 3CL protease-cleaved GSDMD fragments do not induce pyroptosis. A Schematic of GSDMD and its cleaved fragments. B Morphological changes in 293T cells expressing 3 ​× ​Flag-tagged GSDMD fragments. 293T cells were transfected with indicated plasmids, the morphological changes of cells were observed by optical microscope at 24 ​h after transfection. Viability ( C ) and LDH release ( D ) of 293T cells expressing 3 ​× ​Flag-tagged GSDMD fragments. 293T cells were treated as described in panel B, cell viability was assessed using the CCK-8 assay, and cell death was measured by LDH release using an LDH Release Assay Kit. E Western blot analysis of 3 ​× ​Flag-tagged GSDMD fragment expression. 293T cells were treated as described in panel B, cells were lysed and analyzed by Western blotting. Two-tailed Student's t -test was used for two-group comparisons. ∗∗∗, P ​< ​0.001.

Article Snippet: The SARS-CoV-2 3CL protease inhibitor PF-00835231 (HY-137048) was purchased from MedChemExpress ( ).

Techniques: Expressing, Transfection, Microscopy, CCK-8 Assay, Lactate Dehydrogenase Assay, Western Blot, Two Tailed Test

Journal: Virologica Sinica

Article Title: The SARS-CoV-2 3CL protease inhibits pyroptosis through the cleavage of gasdermin D

doi: 10.1016/j.virs.2025.03.006

Figure Lengend Snippet: 3CL protease-cleaved GSDMD fragments do not inhibit SARS-CoV-2 replication. A Western blot analysis of the SARS-CoV-2 N protein in H1299-ACE2 cells expressing GSDMD fragments. H1299-ACE2 cells were transfected with indicated plasmids. At 24 ​h after transfection, cells were infected with SARS-CoV-2 at an MOI of 0.01. After 24 ​h post infection, cells were lysed and analyzed by Western blotting. B–C qRT-PCR analysis of intracellular ( B ) and supernatant ( C ) SARS-CoV-2 RNA levels. H1299-ACE2 cells were treated as described in panel A, the intracellular and supernatant SARS-CoV-2 RNA levels were evaluated by quantitative real-time PCR. Data are expressed as fold change of the viral mRNA level, normalized to GSDMD. D TCID 50 assay of viral titres in supernatants. Two-tailed Student's t -test was used for two-group comparisons. ∗, P ​< ​0.05; ∗∗∗, P ​< ​0.001.

Article Snippet: The SARS-CoV-2 3CL protease inhibitor PF-00835231 (HY-137048) was purchased from MedChemExpress ( ).

Techniques: Western Blot, Expressing, Transfection, Infection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Virologica Sinica

Article Title: The SARS-CoV-2 3CL protease inhibits pyroptosis through the cleavage of gasdermin D

doi: 10.1016/j.virs.2025.03.006

Figure Lengend Snippet: Differences in the GSDMD cleavage sites of viral proteases from various viruses. A–C Western blot analysis of wild-type and mutant HA-mCherry-GSDMD cleavage by 3CL proteases from SARS-CoV ( A ), MERS-CoV ( B ), and 3C protease of EV71 ( C ) in 293T cells. D–E Western blot analysis of GSDMD cleavage by the wild-type and C145A mutant 3CL proteases from SARS-CoV ( D ) and the wild-type and C148A mutant 3CL proteases from MERS-CoV ( E ).

Article Snippet: The SARS-CoV-2 3CL protease inhibitor PF-00835231 (HY-137048) was purchased from MedChemExpress ( ).

Techniques: Western Blot, Mutagenesis