NMPylation and de-NMPylation of SARS-CoV-2 nsp9 by the NiRAN domain

  1. Bing Wang
  2. Dmitri Svetlov
  3. Irina Artsimovitch

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Abstract

The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.

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  1. Version published to 10.1093/nar/gkab677
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    SciScore for 10.1101/2021.06.13.448258: (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.

    Table 2: Resources

    Recombinant DNA
    SentencesResources
    The fusion protein was treated with TEV protease (for pIA1364) or SUMO protease (for pIA1414) at 4 °C overnight.
    pIA1364
    suggested: None
    Software and Algorithms
    SentencesResources
    Multiple sequence alignment (MSA) was done by MAFFT (version 7) (
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    WebLogo (version 3) (25) was used to generate the sequence logos based on the MSA.
    WebLogo
    suggested: (WEBLOGO, RRID:SCR_010236)
    The gels were visualized and quantified using Typhoon FLA9000 (GE Healthcare) and ImageQuant.
    ImageQuant
    suggested: (ImageQuant, RRID:SCR_014246)
    The means and standard deviation (SD) were calculated by Excel (Microsoft).
    Excel
    suggested: None

    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.

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

    About SciScore

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  3. Version published to 10.1101/2021.06.13.448258v1 on bioRxiv