The key features of SARS-CoV-2 leader and NSP1 required for viral escape of NSP1-mediated repression

  1. Lucija Bujanic
  2. Olga Shevchuk
  3. Nicolai von Kügelgen
  4. Anna Kalinina
  5. Katarzyna Ludwik
  6. David Koppstein
  7. Nadja Zerna
  8. Albert Sickmann
  9. Marina Chekulaeva

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Abstract

SARS-CoV-2, responsible for the ongoing global pandemic, must overcome a conundrum faced by all viruses. To achieve its own replication and spread, it simultaneously depends on and subverts cellular mechanisms. At the early stage of infection, SARS-CoV-2 expresses the viral nonstructural protein 1 (NSP1), which inhibits host translation by blocking the mRNA entry tunnel on the ribosome; this interferes with the binding of cellular mRNAs to the ribosome. Viral mRNAs, on the other hand, overcome this blockade. We show that NSP1 enhances expression of mRNAs containing the SARS-CoV-2 leader. The first stem–loop (SL1) in the viral leader is both necessary and sufficient for this enhancement mechanism. Our analysis pinpoints specific residues within SL1 (three cytosine residues at the positions 15, 19, and 20) and another within NSP1 (R124), which are required for viral evasion, and thus might present promising drug targets. We target SL1 with the antisense oligo (ASO) to efficiently and specifically down-regulate SARS-CoV-2 mRNA. Additionally, we carried out analysis of a functional interactome of NSP1 using BioID and identified components of antiviral defense pathways. Our analysis therefore suggests a mechanism by which NSP1 inhibits the expression of host genes while enhancing that of viral RNA. This analysis helps reconcile conflicting reports in the literature regarding the mechanisms by which the virus avoids NSP1 silencing.

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  1. Version published to 10.1261/rna.079086.121
  2. ScreenIT

    SciScore for 10.1101/2021.09.13.460054: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The membrane was probed with the following primary antibodies: mouse anti-flag antibody 1:2000 (F1804 Sigma), mouse anti-beta-actin 1:5000 (A2228 Sigma).
    anti-flag
    suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
    anti-beta-actin
    suggested: (Sigma-Aldrich Cat# A2228, RRID:AB_476697)
    Experimental Models: Cell Lines
    SentencesResources
    For BioID experiments, HEK293T cells were transfected with constructs encoding NSP1-BioID, NSP1-KH164AA-BioID, NSP1-RK124AA-BioID or BioID alone.
    HEK293T
    suggested: None
    Recombinant DNA
    SentencesResources
    DNA constructs: 3xflag-SARS-CoV-2 NSP1-encoding plasmid and pEBG-3xflag, used as a vector, have been described previously (19).
    NSP1-encoding
    suggested: None
    Analogous plasmid expressing SARS-CoV-1 NSP1 was generated using a similar strategy: CDS of NSP1 was PCR amplified, using SARS-CoV-1 cDNA as a template, and cloned between SbfI and NotI sites of pEBG-3xflag.
    pEBG-3xflag
    suggested: None
    For BioID constructs, BioID CDS was PCR amplified from pcDNA 3.1-BioID (17) and cloned between BstXI and NotI of pEBG-sic (21), to produce pEBG-BioID.
    pcDNA 3.1-BioID
    suggested: None
    pEBG-BioID
    suggested: None
    Software and Algorithms
    SentencesResources
    For data analysis, all MS raw data were processed with Proteome Discoverer software 2.3.0.523 (Thermo Scientific, Germany) and searched in a target/decoy fashion against a concatenated version of the human Uniprot database (downloaded on on November 2019, 20300 target sequences); NSP1 from SARS-CoV-2 and BioID SEQUEST-HT algorithm.
    Proteome Discoverer
    suggested: (Proteome Discoverer, RRID:SCR_014477)
    The false discovery rate was set to 0.01 for both peptide and protein identifications using Percolator.
    Percolator
    suggested: (OMSSAPercolator, RRID:SCR_000287)
    Enrichment (log2 fold change) of proteins between pulldown fractions or lysate samples was calculated using a generalised linear model (R limma package, (25)) on imputed log2-transformed LFQ values.
    limma
    suggested: (LIMMA, RRID:SCR_010943)

    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.

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

    About SciScore

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  3. Version published to 10.1101/2021.09.13.460054v1 on bioRxiv