SARS-CoV-2 proteases cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species and the search for reservoir hosts

  1. Mehdi Moustaqil
  2. Emma Ollivier
  3. Hsin-Ping Chiu
  4. Sarah Van Tol
  5. Paulina Rudolffi-Soto
  6. Christian Stevens
  7. Akshay Bhumkar
  8. Dominic J.B. Hunter
  9. Alex Freiberg
  10. David Jacques
  11. Benhur Lee
  12. Emma Sierecki
  13. Yann Gambin

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Abstract

The genome of SARS-CoV-2 (SARS2) encodes for two viral proteases (NSP3/ papain-like protease and NSP5/ 3C-like protease or major protease) that are responsible for cleaving viral polyproteins for successful replication. NSP3 and NSP5 of SARS-CoV (SARS1) are known interferon antagonists. Here, we examined whether the protease function of SARS2 NSP3 and NSP5 target proteins involved in the host innate immune response. We designed a fluorescent based cleavage assay to rapidly screen the protease activity of NSP3 and NSP5 on a library of 71 human innate immune proteins (HIIPs), covering most pathways involved in human innate immunity. By expressing each of these HIIPs with a genetically encoded fluorophore in a cell-free system and titrating in the recombinant protease domain of NSP3 or NSP5, we could readily detect cleavage of cognate HIIPs on SDS-page gels. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type- I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of IL-6 and inflammatory response observed in COVID-19 patients. Surprisingly, both NLRP12 and TAB1 have each two distinct cleavage sites. We demonstrate that in mice, the second cleavage site of NLRP12 is absent. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for in-depth studies into the pathophysiology of COVID-19 and should facilitate the search or development of more effective animal models for severe COVID-19. Finally, we discovered that one particular species of bats, David’s Myotis, possesses the five cleavage sites found in humans for NLRP12, TAB1 and IRF3. These bats are endemic from the Hubei province in China and we discuss its potential role as reservoir for the evolution of SARS1 and SASR2.

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  1. ScreenIT

    SciScore for 10.1101/2020.06.05.135699: (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.

    Table 2: Resources

    Antibodies
    SentencesResources
    The following commercial antibodies were used: anti-IRF3 (ab68481) and anti-ACE2 (ab108252) from Abcam, anti-NLRP12 (PA5-21027) from Invitrogen, anti-TAB1 (#3226) from Cell Signaling, anti-SARS-CoV-2 N (GTX632269) from Genetex, anti- TMPRSS2 (sc-515727) from Santa Cruz Biotechnology, anti-COXIV (11242-1-AP) from Proteintech.
    anti-IRF3
    suggested: (Abcam Cat# ab68481, RRID:AB_11155653)
    anti-ACE2
    suggested: (Abcam Cat# ab108252, RRID:AB_10864415)
    anti-NLRP12
    suggested: (Thermo Fisher Scientific Cat# PA5-21027, RRID:AB_11152858)
    anti-TAB1
    suggested: (Cell Signaling Technology Cat# 3226, RRID:AB_2140247)
    anti-SARS-CoV-2 N (GTX632269
    suggested: None
    anti- TMPRSS2
    suggested: (Santa Cruz Biotechnology Cat# sc-33533, RRID:AB_2205595)
    anti-COXIV
    suggested: (Proteintech Cat# 11242-1-AP, RRID:AB_2085278)
    Recombinant DNA
    SentencesResources
    Alexandrov and sourced from Addgene (Addgene plasmid # 67137; http://n2t.net/addgene:67137; RRID:Addgene_67137).
    detected: RRID:Addgene_67137)

    Results from OddPub: Thank you for sharing your data.

    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 did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 11, 12 and 13. 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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    About SciScore

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  2. Version published to 10.1101/2020.06.05.135699v1 on bioRxiv