Structure–function analysis of the nsp14 N7–guanine methyltransferase reveals an essential role in Betacoronavirus replication

  1. Natacha S. Ogando
  2. Priscila El Kazzi
  3. Jessika C. Zevenhoven-Dobbe
  4. Brenda W. Bontes
  5. Alice Decombe
  6. Clara C. Posthuma
  7. Volker Thiel
  8. Bruno Canard
  9. François Ferron
  10. Etienne Decroly
  11. Eric J. Snijder

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Abstract

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic emphasizes the urgent need to develop efficient broad-spectrum anti-CoV drugs. The structure–function characterization of conserved CoV replicative enzymes is key to identifying the most suitable drug targets. Using a multidisciplinary comparative approach and different betacoronaviruses, we characterized the key conserved residues of the nsp14 (N7-guanine)–methyltransferase, a poorly defined subunit of the CoV messenger RNA–synthesizing machinery. Our study highlights the unique structural features of this enzyme and establishes its essential role in betacoronavirus replication, while identifying two residues that are critical for the replication of the four betacoronaviruses tested, including SARS-CoV-2.

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  1. Version published to 10.1073/pnas.2108709118
  2. ScreenIT

    SciScore for 10.1101/2021.05.17.444407: (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

    Experimental Models: Cell Lines
    SentencesResources
    In order to amplify viral progeny and titrate recombinant CoVs by plaque assay, Vero E6 cells were used for SARS-CoV and SARS-CoV-2, HuH7 cells for MERS-CoV, and 17Cl1 cells for MHV.
    HuH7
    suggested: None
    Transfected BHK-21 cells were mixed in a 1:1 ratio with cells susceptible to CoV infection: Vero E6 cells (for SARS-CoV and SARS-CoV-2), HuH7 cells for MERS-CoV, or 17Cl1 cells (for MHV).
    BHK-21
    suggested: None
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    Bioinformatics analysis: Forty-seven CoV nsp14 sequences were retrieved (a complete list is provided in Table S1) and aligned using MAFFT.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    Delineation of motif I to VI was done manually using Seaview and WebLogo (74,
    Seaview
    suggested: (SeaView, RRID:SCR_015059)
    WebLogo
    suggested: (WEBLOGO, RRID:SCR_010236)
    In vitro nsp14 N7-MTase activity assay: Reaction mixtures contained 50 or 200 nM of SARS-CoV or MERS-CoV recombinant nsp14, 7 nM GpppACCCC synthetic RNA substrate, 40 mM Tris-HCl (pH 8.0), 10 mM DTT, 5 mM MgCl2, 1.9 μM SAM, 0.1 μM 3H-SAM (Perkin Elmer).
    SAM
    suggested: (SAM, RRID:SCR_010951)

    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.

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

    About SciScore

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  3. Version published to 10.1101/2021.05.17.444407v2 on bioRxiv
  4. Version published to 10.1101/2021.05.17.444407v1 on bioRxiv