Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2

  1. Matthias Thoms
  2. Robert Buschauer
  3. Michael Ameismeier
  4. Lennart Koepke
  5. Timo Denk
  6. Maximilian Hirschenberger
  7. Hanna Kratzat
  8. Manuel Hayn
  9. Timur Mackens-Kiani
  10. Jingdong Cheng
  11. Christina M. Stürzel
  12. Thomas Fröhlich
  13. Otto Berninghausen
  14. Thomas Becker
  15. Frank Kirchhoff
  16. Konstantin M.J. Sparrer
  17. Roland Beckmann

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Abstract

SARS-CoV-2 is the causative agent of the current COVID-19 pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1) which suppresses host gene expression by ribosome association via an unknown mechanism. Here, we show that Nsp1 from SARS-CoV-2 binds to 40S and 80S ribosomes, resulting in shutdown of capped mRNA translation both in vitro and in cells. Structural analysis by cryo-electron microscopy (cryo-EM) of in vitro reconstituted Nsp1-40S and of native human Nsp1-ribosome complexes revealed that the Nsp1 C-terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks RIG-I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.

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  1. ScreenIT

    SciScore for 10.1101/2020.05.18.102467: (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

    No key resources detected.

    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: 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 10.1101/2020.05.18.102467: (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
    Western blot analysis of the indicated gradient fractions stained with anti-FLAG antibody are shown below and separate blots are indicated with dashed lines .
    anti-FLAG
    suggested: None
    The translation product was analyzed by western blot analysis using a anti-V5 antibody ( upper panel) .
    anti-V5
    suggested: None
    Western blot of whole cell lysates ( WCL ) stained with anti-V5 and anti-actin antibodies ( bottom panel) .
    anti-actin
    suggested: None
    Immunoblot of whole cell lysates stained with anti-V5 , anti-FLAG and anti-GAPDH antibodies ( bottom panel) .
    anti-V5 ,
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>anti-GAPDH</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immunoblot of whole cell lysates stained with anti-V5 , anti-FLAG and anti-GAPDH antibodies ( bottom panel) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-V5 ,</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-GAPDH</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">( C ) Polyribosome gradient analysis of HEK293T cell lysate ( Control ) and lysate from HEK293T cells transiently transfected with N-terminal 3xFLAG-3C tagged Nsp1 and Nsp1-mt constructs from SCoV-1 and SCoV-2 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK293T</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">( D ) Cell-free in vitro translation of a capped reporter mRNA with rabbit reticulocytes ( RRL ) and HeLa S3 lysate.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HeLa S3</b></div>
        <div>suggested: CLS Cat# 300384/p699_HeLa_S3, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0058">CVCL_0058</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Measles virus ( MeV ) V protein , which inhibits immune sensors like MDA5 ( 41) , but has little effect on RIG-I-dependent signaling , was included as control for specific induction of RIG-I-dependent signaling .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>MDA5</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Huang et al. , Alphacoronavirus transmissible gastroenteritis virus nsp1 protein suppresses protein translation in mammalian cells and in cell-free HeLa cell extracts but not in rabbit reticulocyte lysate.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HeLa</b></div>
        <div>suggested: CLS Cat# 300194/p772_HeLa, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0030">CVCL_0030</a></div>
      </div>
    </td></tr></table>

    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 OddPub: Thank you for sharing your data.

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2020.05.18.102467 on bioRxiv