The nonstructural protein 5 of coronaviruses antagonizes GSDMD-mediated pyroptosis by cleaving and inactivating its pore-forming p30 fragment

  1. Fushan Shi
  2. Qian Lv
  3. Tingjun Wang
  4. Jidong Xu
  5. Wei Xu
  6. Yuhua Shi
  7. Xinyu Fu
  8. Tianming Yang
  9. Yang Yang
  10. Lenan Zhuang
  11. Weihuan Fang
  12. Jinyan Gu
  13. Xiaoliang Li

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Abstract

Coronaviruses (CoV) are a family of RNA viruses that typically cause respiratory, enteric and hepatic diseases in animals and humans. Here, we used porcine epidemic diarrhea virus (PEDV) as a model of coronaviruses (CoVs) to illustrate the reciprocal regulation between CoVs infection and pyroptosis. For the first time, we clarified the molecular mechanism of porcine Gasdermin D (pGSDMD)-mediated pyroptosis and demonstrated that amino acids T239 and F240 within pGSDMD-p30 are critical for pyroptosis. Furthermore, 3C-like protease Nsp5 from SARS-CoV-2, MERS-CoV, PDCoV and PEDV can cleave human/porcine GSDMD at the Q193-G194 junction upstream of the caspase-1 cleavage site to produce two fragments which fail to trigger pyroptosis or inhibit viral replication. Thus, we provide clear evidence that coronoviruses may utilize viral Nsp5-GSDMD pathway to help their host cells escaping from pyroptosis, protecting the replication of the virus during the initial period, which suggest an important strategy for coronoviruses infection and sustain.

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  1. ScreenIT

    SciScore for 10.1101/2021.02.23.432418: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Anti-Flag antibody (F1804), anti-MYC antibody (C3956) and anti-GSDMD antibody (G7422) were purchased from Sigma.
    Anti-Flag
    suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
    F1804
    suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
    anti-MYC
    suggested: None
    C3956
    suggested: (Sigma-Aldrich Cat# C3956, RRID:AB_439680)
    Anti-HA antibody (3724) was purchased from Cell Signaling Technology.
    Anti-HA
    suggested: None
    Anti-β-actin antibody (A01010) was purchased from Abbkine.
    Anti-β-actin
    suggested: (Abbkine Cat# A01010, RRID:AB_2737288)
    Anti-GSDMDC1 antibody (sc-393581) was purchased from Santa Cruz.
    Anti-GSDMDC1
    suggested: (Santa Cruz Biotechnology Cat# sc-393581, RRID:AB_2819179)
    The anti-PEDV N monoclonal antibody and the anti-GSDMD polyclonal antibody were prepared in our laboratory as previously described28,29. Necrosulfonamide (S8251) and Disulfiram (S1680) were purchased from Selleck.
    anti-PEDV N
    suggested: None
    anti-GSDMD
    suggested: None
    S1680
    suggested: (Abcam Cat# S1680, RRID:AB_10637724)
    The membranes were blocked with QuickBlock Blocking Buffer for Western blotting (Beyotime, P0252) for 1 h and then incubated with primary antibodies diluted with QuickBlock Primary Antibody Dilution Buffer for Western blotting (Beyotime, P0256) at 4□ overnight.
    P0256
    suggested: None
    After washing 3 times with PBS (3 min each), the cells were incubated with the secondary antibody (Goat Anti-Rabbit IgG Alexa Fluor 568, Abcam, ab175471) at 37 □ for 60 min in the dark and then they were washed 3 times with PBS (5 min each).
    Anti-Rabbit IgG
    suggested: (Abcam Cat# ab175471, RRID:AB_2576207)
    Experimental Models: Cell Lines
    SentencesResources
    Cells and virus: African green monkey kidney cells (Vero cells) and Human embryonic kidney 293T cells (HEK293T) were cultured in DMEM (Hyclone, SH30243.01) containing 10% fetal bovine serum (FBS) (Corille, C1015-05) and 5% Penicillin-Streptomycin Solution (Hyclone, SV30010)
    Vero
    suggested: None
    293T
    suggested: None
    IPEC-J2 cells were maintained in DMEM/F12 (Hyclone, SH30023.01) supplemented with 10% FBS and 5% Penicillin-Streptomycin Solution.
    IPEC-J2
    suggested: None
    Confocal immunofluorescence assay: HEK293T cells were seeded in 24-well plates on coverslips and after cultured overnight indicated plasmids were transfected.
    HEK293T
    suggested: None
    Software and Algorithms
    SentencesResources
    SnapGene software were used to perform the multiple-sequence alignment.
    SnapGene
    suggested: (SnapGene, RRID:SCR_015052)
    Data are presented as mean ± SD and analyzed by the two-tailed Student’s t test or one-way ANOVA followed by Tukey’s multiple comparisons test by Prism software (GraphPad).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 26 and 27. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.02.23.432418v1 on bioRxiv