Crystal structures and fragment screening of SARS-CoV-2 NSP14 reveal details of exoribonuclease activation and mRNA capping and provide starting points for antiviral drug development

  1. Nergis Imprachim
  2. Yuliana Yosaatmadja
  3. Joseph A Newman

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Abstract

NSP14 is a dual function enzyme containing an N-terminal exonuclease domain (ExoN) and C-terminal Guanine-N7-methyltransferase (N7-MTase) domain. Both activities are essential for the viral life cycle and may be targeted for anti-viral therapeutics. NSP14 forms a complex with NSP10, and this interaction enhances the nuclease but not the methyltransferase activity. We have determined the structure of SARS-CoV-2 NSP14 in the absence of NSP10 to 1.7 Å resolution. Comparisons with NSP14/NSP10 complexes reveal significant conformational changes that occur within the NSP14 ExoN domain upon binding of NSP10, including helix to coil transitions that facilitate the formation of the ExoN active site and provide an explanation of the stimulation of nuclease activity by NSP10. We have determined the structure of NSP14 in complex with cap analogue 7MeGpppG, and observe conformational changes within a SAM/SAH interacting loop that plays a key role in viral mRNA capping offering new insights into MTase activity. We perform an X-ray fragment screen on NSP14, revealing 72 hits bound to sites of inhibition in the ExoN and MTase domains. These fragments serve as excellent starting point tools for structure guided development of NSP14 inhibitors that may be used to treat COVID-19 and potentially other future viral threats.

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  1. Version published to 10.1093/nar/gkac1207
  2. ScreenIT

    SciScore for 10.1101/2022.03.11.483836: (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
    Cloning and expression of NSP14: The plasmid for NSP14 with His6 and Z-Basic tags at the N-terminal was synthesized in a pNIC-ZB vector with codon optimization for expression in Escherichia coli (Supplementary Table 1).
    pNIC-ZB
    suggested: RRID:Addgene_26107)
    Software and Algorithms
    SentencesResources
    Refinement was performed using PHENIX REFINE35.
    PHENIX
    suggested: (Phenix, RRID:SCR_014224)
    Refinement was performed using BUSTER.
    BUSTER
    suggested: (BUSTER, RRID:SCR_015653)

    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 10.1101/2022.03.11.483836v1 on bioRxiv