SARS-CoV-2 Nsp1 N-terminal and linker regions as a platform for host translational shutoff

  1. Andrea Graziadei
  2. Fabian Schildhauer
  3. Christian Spahn
  4. Matthew Kraushar
  5. Juri Rappsilber

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Abstract

In the early stages of SARS-CoV-2 infection, non-structural protein 1 (Nsp1) inhibits the innate immune response by inserting its C-terminal helices into the mRNA entry channel of the ribosome and promoting mRNA degradation. Nevertheless, the mechanism by which Nsp1 achieves host translational shutoff while allowing for viral protein synthesis remains elusive. We set out to characterize the interactome of full-length Nsp1 and its topology by crosslinking mass spectrometry in order to investigate the role of the N-terminal domain and linker regions in host translational shutoff. We find that these regions are in contact with 40S proteins lining the mRNA entry channel and detect a novel interaction with the G subunit of the eIF3 complex. The crosslink-derived distance restraints allowed us to derive an integrative model of full-length Nsp1 on the 40S subunit, reporting on the dynamic interface between Nsp1, the ribosome and the eIF3 complex. The significance of the Nsp1-eIF3G interaction is supported by further evidence that Nsp1 predominantly binds to 40-43S complexes. Our results point towards a mechanism by which Nsp1 is preferentially recruited to canonical initiation complexes, leading to subsequent mRNA degradation.

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  1. Version published to 10.1101/2022.02.10.479924v2 on bioRxiv
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    SciScore for 10.1101/2022.02.10.479924: (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

    Experimental Models: Cell Lines
    SentencesResources
    HEK293T cells were transfected with pcDNA5_FRT_TO-3xFLAG-3C-Nsp1 (prof.
    HEK293T
    suggested: None
    Recombinant DNA
    SentencesResources
    HEK293T cells were transfected with pcDNA5_FRT_TO-3xFLAG-3C-Nsp1 (prof.
    pcDNA5_FRT_TO-3xFLAG-3C-Nsp1
    suggested: None
    Each replica of HEK293T cells transfected with either pcDNA5_FRT_TO-3xFLAG-3C-Nsp1 or pcDNA3 (control) were trypsinized and collected by centrifugation and washed twice with phosphate-buffered saline (PBS, Corning).
    pcDNA3
    suggested: RRID:Addgene_15475)
    Sucrose gradient analysis: Sucrose density gradient ultracentrifugation fractionation: HEK293T cells were transfected with pcDNA5_FRT_TO-3xFLAG-3C-Nsp1 or with pEGFP-C1 in triplicates as described above.
    pEGFP-C1
    suggested: None
    Software and Algorithms
    SentencesResources
    Transfection of HEK293T cells was performed by mixing plasmid DNA with PolyJet™ In Vitro DNA Transfection Reagent (SignaGen Laboratories) and incubation with cells for 3 h.
    PolyJet™
    suggested: None
    LC-MS protein identification of crosslinked samples and analysis: LC-MS/MS analysis of DSSO crosslinked sample was performed on an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific, Germany) connected to an Ultimate 3000 RSLCnano system (Dionex, Thermo Fisher Scientific, Germany), which were operated under Tune 3.4, SII for Xcalibur 1.6 and Xcalibur 4.4.
    Xcalibur
    suggested: (Thermo Xcalibur, RRID:SCR_014593)
    AP-MS analysis: Raw data from mass spectrometry were searched as described above using MaxQuant (1.6.17.).
    MaxQuant
    suggested: (MaxQuant, RRID:SCR_014485)
    The domain was built in MODELLER version 9.23 (41) and fitted in the density for “protein X” in the 43S preinitiation complex.
    MODELLER
    suggested: (MODELLER, RRID:SCR_008395)

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

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  3. Version published to 10.1101/2022.02.10.479924v1 on bioRxiv