Suppression of MDA5-mediated antiviral immune responses by NSP8 of SARS-CoV-2

  1. Ziwei Yang
  2. Xiaolin Zhang
  3. Fan Wang
  4. Peihui Wang
  5. Ersheng Kuang
  6. Xiaojuan Li

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Abstract

Melanoma differentiation-associated gene-5 (MDA5) acts as a cytoplasmic RNA sensor to detect viral dsRNA and mediates type I interferon (IFN) signaling and antiviral innate immune responses to infection by RNA viruses. Upon recognition of viral dsRNA, MDA5 is activated with K63-linked polyubiquitination and then triggers the recruitment of MAVS and activation of TBK1 and IKK, subsequently leading to IRF3 and NF-κB phosphorylation. Great numbers of symptomatic and severe infections of SARS-CoV-2 are spreading worldwide, and the poor efficacy of treatment with type I interferon and antiviral agents indicates that SARS-CoV-2 escapes from antiviral immune responses via an unknown mechanism. Here, we report that SARS-CoV-2 nonstructural protein 8 (NSP8) acts as an innate immune suppressor and inhibits type I IFN signaling to promote infection of RNA viruses. It downregulates the expression of type I IFNs, IFN-stimulated genes and proinflammatory cytokines by binding to MDA5 and impairing its K63-linked polyubiquitination. Our findings reveal that NSP8 mediates innate immune evasion during SARS-CoV-2 infection and may serve as a potential target for future therapeutics for SARS-CoV-2 infectious diseases.

Importance

The large-scale spread of COVID-19 is causing mass casualties worldwide, and the failure of antiviral immune treatment suggests immune evasion. It has been reported that several nonstructural proteins of severe coronaviruses suppress antiviral immune responses; however, the immune suppression mechanism of SARS-CoV-2 remains unknown. Here, we revealed that NSP8 protein of SARS-CoV-2 directly blocks the activation of the cytosolic viral dsRNA sensor MDA5 and significantly downregulates antiviral immune responses. Our study contributes to our understanding of the direct immune evasion mechanism of SARS-CoV-2 by showing that NSP8 suppresses the most upstream sensor of innate immune responses involved in the recognition of viral dsRNA.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.12.247767: (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
    The following antibodies were used in this study: goat anti-mouse IRDye680RD (C90710-09) and goat anti-rabbit IRDye800CW (C80925-05), which were purchased from Li-COR; anti-HA (M132-3), purchased from MBL; anti-Flag (AE005), anti-GFP (AE0122) and anti-IRF3 (A2172), purchased from ABclonal; anti-β-actin (HC201-01), purchased from TransGen; and anti-pTBK1 (#5483P), anti-TBK1 (#3504), anti-pIRF3 (#4947), anti-phospho-NF-κB p65 (#3033), and anti-NF-κB p65 (#8242), purchased from Cell Signaling Technology. Plasmids: The following plasmids were used.
    anti-mouse
    suggested: (Creative Biomart Cat# CAB-3033MH, RRID:AB_10963325)
    anti-rabbit
    suggested: (Zyagen Cat# NEU-4947, RRID:AB_10699970)
    anti-HA
    suggested: None
    M132-3
    suggested: (MBL International Cat# M132-3 ??, RRID:AB_843156)
    anti-Flag (AE005)
    suggested: None
    anti-GFP
    suggested: None
    anti-IRF3
    suggested: (ABclonal Cat# A2172, RRID:AB_2764190)
    A2172
    suggested: (Sigma-Aldrich Cat# A2172, RRID:AB_476695)
    anti-β-actin (HC201-01)
    suggested: (Transgen Biotech Cat# HC201, RRID:AB_2860007)
    anti-pTBK1
    suggested: None
    anti-TBK1
    suggested: (Cell Signaling Technology Cat# 3504, RRID:AB_2255663)
    anti-pIRF3
    suggested: None
    anti-phospho-NF-κB p65
    suggested: None
    anti-NF-κB p65
    suggested: (Proteintech Cat# 10745-1-AP, RRID:AB_2178878)
    #8242
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    A549 cells (human adenocarcinoma lung tissue-derived epithelial cells) were cultured in RPMI 1640 (Gibco) medium containing 10% FBS.
    A549
    suggested: None
    Transfection and luciferase reporter assays: HEK293T cells were seeded in 24-well plates overnight and then transfected using Lipofectamine 2000 (Invitrogen) with 100 ng ISRE luciferase reporter (firefly luciferase), 20 ng pRL-TK plasmid (Renilla luciferase), 150 ng Flag-MDA5 expressing plasmid and increasing amounts (0, 100, or 200 ng) of NSP8-expressing plasmid.
    HEK293T
    suggested: None
    MDA5-CARDs with NSP8.pdb and MDA5-CARDs with K63-Ub.pdb were the best fit prediction models chosen from the results.
    MDA5-CARDs
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Virus infection: VSV-eGFP was kindly provided by Dr Meng Lin, School of Life Sciences, Sun Yat-Sen University.
    VSV-eGFP
    suggested: None
    MDA5-CARDs.pdb was input into ZDOCK-SERVER 28 as a receptor, while NSP8 or K63-Ub was input as a ligand for docking computation.
    MDA5-CARDs
    suggested: None
    Software and Algorithms
    SentencesResources
    All the pdb files were processed and visualized with PyMOL (Schrödinger.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)

    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: 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.

  2. ScreenIT

    SciScore for 10.1101/2020.08.12.247767: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    The following antibodies were used in this study: goat anti-mouse IRDye680RD (C90710-09) and goat anti-rabbit IRDye800CW (C80925-05), which were purchased from Li-COR; anti-HA (M132-3), purchased from MBL; anti-Flag (AE005), anti-GFP (AE0122) and anti-IRF3 (A2172), purchased from ABclonal; anti-β-actin (HC201-01), purchased from TransGen; and anti-pTBK1 (#5483P), anti-TBK1 (#3504), anti-pIRF3 (#4947), anti-phospho-NF-κB p65 (#3033), and anti-NF-κB p65 (#8242), purchased from Cell Signaling Technology.
    anti-mouse
    suggested: (Creative Biomart Cat# CAB-3033MH, RRID:AB_10963325)
    anti-rabbit
    suggested: (Zyagen Cat# NEU-4947, RRID:AB_10699970)
    anti-HA
    suggested: None
    M132-3
    suggested: (MBL International Cat# M132-3 ??, RRID:AB_843156)
    anti-Flag (AE005)
    suggested: None
    anti-IRF3
    suggested: (ABclonal Cat# A2172, RRID:AB_2764190)
    A2172
    suggested: (Sigma-Aldrich Cat# A2172, RRID:AB_476695)
    anti-β-actin (HC201-01)
    suggested: (Transgen Biotech Cat# HC201, RRID:AB_2860007)
    anti-pTBK1
    suggested: None
    anti-TBK1
    suggested: (Cell Signaling Technology Cat# 3504, RRID:AB_2255663)
    anti-pIRF3
    suggested: None
    anti-phospho-NF-κB p65
    suggested: None
    anti-NF-κB p65
    suggested: None
    #8242
    suggested: None
    Then, cell lysates were subjected to coimmunoprecipitation using anti-GFP beads, followed by immunoblotting with the indicated antibodies. c.
    anti-GFP
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    293T cells were transfected with empty vector or NSP-expressing plasmids for 24 h.
    293T
    suggested: None
    MDA5-CARDs with NSP8.pdb and MDA5-CARDs with K63-Ub.pdb were the best fit prediction models chosen from the results.
    MDA5-CARDs
    suggested: None
    HEK293T cells were cotransfected with mCherry-NSP8 and GFP-MDA5.
    HEK293T
    suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
    Cell lysates were subjected to coimmunoprecipitation using anti-GFP beads, followed by immunoblotting analysis with the indicated antibodies. a. A549 cells were transfected with an empty vector or Flag-NSP8.
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    Experimental Models: Organisms/Strains
    SentencesResources
    MDA5-CARDs.pdb was input into ZDOCK-SERVER 28 as a receptor, while NSP8 or K63-Ub was input as a ligand for docking computation.
    MDA5-CARDs
    suggested: None
    Software and Algorithms
    SentencesResources
    Predicted docking models were processed in PyMOL for visualization.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)

    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 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: We did not find any issues relating to colormaps.

    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.

  3. Version published to 10.1101/2020.08.12.247767v1 on bioRxiv