SARS‐CoV‐2 ORF9b antagonizes type I and III interferons by targeting multiple components of the RIG‐I/MDA‐5–MAVS, TLR3–TRIF, and cGAS–STING signaling pathways

  1. Lulu Han
  2. Meng‐Wei Zhuang
  3. Jian Deng
  4. Yi Zheng
  5. Jing Zhang
  6. Mei‐Ling Nan
  7. Xue‐Jing Zhang
  8. Chengjiang Gao
  9. Pei‐Hui Wang

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Abstract

The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) contributes to the pathogenesis of coronavirus disease 2019 (COVID‐19). The strategy used by SARS‐CoV‐2 to evade antiviral immunity needs further investigation. Here, we reported that SARS‐CoV‐2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS‐CoV‐2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS‐CoV‐2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA‐sensing pathways of RIG‐I/MDA5‐MAVS signaling, including RIG‐I, MDA‐5, MAVS, TBK1, and IKKε, rather than IRF3‐5D, which is the active form of IRF3. SARS‐CoV‐2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA‐sensing pathway of TLR3‐TRIF signaling and the adaptor protein of the cytosolic DNA‐sensing pathway of cGAS–STING signaling, respectively. A mechanistic analysis revealed that the SARS‐CoV‐2 ORF9b protein interacted with RIG‐I, MDA‐5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS‐CoV‐2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS‐CoV‐2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS‐CoV‐2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID‐19.

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  1. Version published to 10.1002/jmv.27050
  2. ScreenIT

    SciScore for 10.1101/2020.08.16.252973: (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
    Rabbit anti-Myc-tag (71D10), rabbit anti-IRF3 (D83B9), rabbit anti-pIRF3 (4D46), rabbit anti-TBK1 (3031S), rabbit anti-pTBK1 (D52C2) were purchased from Cell Signaling Technology; Mouse anti-MAVS was purchased from Santa Cruz; Mouse anti-actin, mouse anti-V5-tag, and rabbit anti-calnexin were purchased from proteintech; Mouse anti-Flag M2 was purchased from Sigma Aldrich; Mouse anti-Myc-tag (9E10) was purchased from Origene; Rabbit anti-GM130 was purchased from Abcam; Rabbit anti-Tom20 antibody was purchased from Abclonal; Mouse anti-HA was purchased from MDL biotech.
    anti-Myc-tag
    suggested: (Cell Signaling Technology Cat# 2278, RRID:AB_490778)
    anti-IRF3
    suggested: None
    anti-pIRF3
    suggested: None
    anti-TBK1
    suggested: (Cell Signaling Technology Cat# 14590, RRID:AB_2798527)
    anti-pTBK1 (D52C2)
    suggested: None
    anti-MAVS
    suggested: None
    anti-actin
    suggested: None
    anti-V5-tag
    suggested: None
    anti-calnexin
    suggested: None
    anti-Flag
    suggested: None
    anti-GM130
    suggested: None
    anti-Tom20
    suggested: None
    anti-HA
    suggested: None
    Supernatants were transferred into new tubes after centrifugation for 10 min at 14,000g and further incubated with the indicated antibodies for 3 hours at 4 □ followed by the addition of protein A/G beads (Santa Cruz), or with Anti-Flag magnetic beads (Bimake), anti-Myc magnetic beads (Bimake).
    anti-Myc
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Constructs and plasmids: Plasmids expressing RIG-I, RIG-IN, MDA-5, MAVS, TBK1, IKKε, IRF3-5D, TRIF, and STING were cloned into mammalian expression vectors and the luciferase reporter plasmids including pGL3-IFN-β-Luc (IFN-β luciferase reporter) and pGL3-IFN-λ1-Luc (IFN-λ1 luciferase reporter) were constructed by inserting the promoter region into pGL3-Basic (Promega, USA) by standard molecular cloning methods as described in our previous publications.
    MDA-5
    suggested: None
    Cell culture: HEK-293, HEK-293T, HeLa, and Vero E6 cells were obtained from the American Type Culture Collection (ATCC), and cultured according to the culture method provide by ATCC.
    HEK-293
    suggested: None
    HeLa
    suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
    Vero E6
    suggested: RRID:CVCL_XD71)
    The luciferase reporter plasmids and the gene expression plasmids were co-transfected into HEK-293T cells as indicated in each figure legend.
    HEK-293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Viral infection: VSV-enhanced green fluorescent protein (eGFP) and SeV were used to infect HeLa, HEK293, or HEK293T cells as described in our previous publications.30-32 Briefly, before infection, prewarmed serum-free DMEM medium at 37°C was used to wash the target cells, after which the virus was diluted to the desired multiplicity of infection (MOI) in serum-free DMEM and incubated with the target cells for 1-2 hours.
    HEK293
    suggested: None
    Immunoblot analysis and immunoprecipitation: For Co-IP assay, HEK293T cells were first transfected with the indicated plasmids for 24 h and further lysed in lysis buffer [1.0% (v/v) NP-40, 50 mM Tris-HCl, pH 7.4, 50 mM EDTA, 0.15 M NaCl] complemented with a protease inhibitor cocktail (Sigma), and a phosphatase inhibitor cocktail (Sigma).
    HEK293T
    suggested: None
    Plaque assays: Vero-E6 cells were used to perform plaque assays to determine the titer of VSV-eGFP.
    Vero-E6
    suggested: None
    Simply, Vero cells at approximately 100% confluency cultured in 24-well plates were infected with serial dilutions of VSV-eGFP.
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    34 Statistical analysis: Statistical analysis was performed using two-tailed unpaired Student’s t-tests by GraphPad Prism 8.0 and Microsoft Excel.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)

    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.

  3. Version published to 10.1101/2020.08.16.252973 on bioRxiv