Highly pathogenic coronavirus N protein aggravates inflammation by MASP-2-mediated lectin complement pathway overactivation

  1. Ting Gao
  2. Lin Zhu
  3. Hainan Liu
  4. Xiaopeng Zhang
  5. Tingting Wang
  6. Yangbo Fu
  7. Hongzhen Li
  8. Qincai Dong
  9. Yong Hu
  10. Zhang Zhang
  11. Jing Jin
  12. Zijing Liu
  13. Weihong Yang
  14. Yaoning Liu
  15. Yanwen Jin
  16. Kaitong Li
  17. Yongjiu Xiao
  18. Junli Liu
  19. Huailong Zhao
  20. Yue Liu
  21. Ping Li
  22. Jibo Song
  23. Lu Zhang
  24. Yuwei Gao
  25. Sisi Kang
  26. Shoudeng Chen
  27. Qingjun Ma
  28. Xiuwu Bian
  29. Wei Chen
  30. Xuan Liu
  31. Qing Mao
  32. Cheng Cao

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Abstract

Excessive inflammatory responses contribute to the pathogenesis and lethality of highly pathogenic human coronaviruses, but the underlying mechanism remains unclear. In this study, the N proteins of highly pathogenic human coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were found to bind MASP-2, a key serine protease in the lectin pathway of complement activation, resulting in excessive complement activation by potentiating MBL-dependent MASP-2 activation, and the deposition of MASP-2, C4b, activated C3 and C5b-9. Aggravated inflammatory lung injury was observed in mice infected with adenovirus expressing the N protein. Complement hyperactivation was also observed in SARS-CoV-2-infected patients. Either blocking the N protein:MASP-2 interaction, MASP-2 depletion or suppressing complement activation can significantly alleviate N protein-induced complement hyperactivation and lung injury in vitro and in vivo. Altogether, these data suggested that complement suppression may represent a novel therapeutic approach for pneumonia induced by these highly pathogenic coronaviruses.

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  1. Version published to 10.1038/s41392-022-01133-5
  2. Version published to 10.1101/2020.03.29.20041962v3 on medRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.03.29.20041962: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: This study was performed with the approval of the ethics committee at the Beijing Institute of Biotechnology and conformed to the relevant regulatory standards.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableSera from 12 mild patients (6 males and 6 females), with an average of 56±12.1 years old and an average 26.3±9.1 days of illness, and sera from 18 severe patients (7 males and 11 females), with an average of 72.4±10.1 years old and an average 40.15±12.74 days of illness, were assayed.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    HRP-conjugated anti-Myc (Santa Cruz), and goat anti-mouse immunoglobulin G (IgG) (Amersham/Pharmacia) antibodies.
    HRP-conjugated anti-Myc ( Santa Cruz)
    suggested: None
    goat anti-mouse immunoglobulin G ( IgG ) ( Amersham/Pharmacia ) antibodies
    suggested: None
    anti-mouse immunoglobulin G ( IgG
    suggested: None
    The cleavage was followed by SDS-PAGE under reducing conditions, and the C4alpha’ fragments, which are left by C4a release after C4 cleavage and can indicate C4 activation, and MASP-2 were detected by immunoblot analysis with anti-C4alpha chain antibody (Santa Cruz) or anti-Flag antibody (Sigma).
    anti-C4alpha chain antibody ( Santa Cruz )
    suggested: None
    anti-Flag
    suggested: None
    Detection of C4, activated C3, and C5b-9 was performed using anti-C4α chain antibody (Santa Cruz)
    C4
    suggested: None
    anti-C4α chain antibody ( Santa Cruz)
    suggested: None
    anti-activated C3 antibody (Santa Cruz), and anti-C5b-9 antibody (Calbiochem), respectively
    anti-activated C3
    suggested: None
    anti-C5b-9
    suggested: None
    Antibody binding was detected using HRP-conjugated sheep anti-mouse antibody or donkey anti-rabbit antibody (R&D).
    anti-mouse
    suggested: None
    anti-rabbit
    suggested: None
    The plates were washed after 32 hr of incubation at 4°C, and the binding of MASP-2 were detected with anti-MASP-2 antibody followed by HRP-conjugated rabbit anti-goat antibody.
    anti-MASP-2
    suggested: None
    anti-goat
    suggested: None
    anti-N monoclonal antibody (200 μg/kg, Sino Biolgical) or C1INH (4 mg/kg, Calbiochem) was injected via the tail vein 30 min before LPS injection.
    anti-N
    suggested: None
    C1INH
    suggested: None
    C3 (Santa Cruz) or C5b-9 (Calbiochem) antibodies as described in the instruction manual.
    C3
    suggested: None
    C5b-9 ( Calbiochem )
    suggested: None
    Anti-C5a antibody therapy: BDB-001 is a mouse-human chimeric (IgG4) antibody against human C5a.
    mouse-human chimeric ( IgG4
    suggested: None
    human C5a
    suggested: None
    Administration of the antibody to human was also approved by the Ethics Committee in Wuhan Huoshenshan Hospital. 300 mg anti-C5a antibody in 250 ml saline was administrated i.v. on day 1, 2, 3, 5, 7, 9, 11 and 13.
    anti-C5a
    suggested: None
    on day 1 , 2 , 3 , 5 , 7 , 9
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture and transfections: The 293T cell line was obtained from the Cell Resource Center of Peking Union Medical College.
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    The IFX-1 cell line has originally been developed by InflaRx GmbH, Germany and was licensed to Beijing Deferengrei Biotech Co., Ltd., a wholly owned subsidiary of Staidson Biopharmaceutical Co., Ltd.
    IFX-1
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Animal experiments: Groups of BALB/c mice were provided from the experimental animal center of the Academy of Military Medical Sciences.
    BALB/c
    suggested: RRID:IMSR_ORNL:BALB/cRl)

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

    Results from JetFighter: We did not find any issues relating to colormaps.

    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.

  4. Version published to 10.1101/2020.03.29.20041962v2 on medRxiv
  5. Version published to 10.1101/2020.03.29.20041962v1 on medRxiv