Characterisation of the SARS-CoV-2 ExoN (nsp14 ExoN -nsp10) complex: implications for its role in viral genome stability and inhibitor identification

  1. Hannah T. Baddock
  2. Sanja Brolih
  3. Yuliana Yosaatmadja
  4. Malitha Ratnaweera
  5. Marcin Bielinski
  6. Lonnie P. Swift
  7. Abimael Cruz-Migoni
  8. Garrett M. Morris
  9. Christopher J. Schofield
  10. Opher Gileadi
  11. Peter J. McHugh

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Abstract

The SARS-CoV-2 coronavirus (CoV) causes COVID-19, a current global pandemic. SARS-CoV-2 belongs to an order of Nidovirales with very large RNA genomes. It is proposed that the fidelity of CoV genome replication is aided by an RNA nuclease complex, formed of non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Here, we confirm that the SARS-CoV-2 nsp14-nsp10 complex is an RNase. Detailed functional characterisation reveals nsp14-nsp10 is a highly versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3’-terminus. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading and purges the nascent replicating RNA strand of a range of potentially replication terminating aberrations. Using the developed assays, we identify a series of drug and drug-like molecules that potently inhibit nsp14-nsp10, including the known Sars-Cov-2 major protease (M pro ) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for bifunctional inhibitors in the treatment of COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.13.248211: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    The protein was generated by transformation into E. coli BL21 Rosetta2 cells and expression in Terrific Broth media supplemented with 30 μg mL-1 kanamycin and 10 mM ZnCl2.
    Rosetta2
    suggested: None
    Software and Algorithms
    SentencesResources
    Gel images collected on a Typhoon scanner were analysed in Image J to determine the amount of substrate remaining in the well (undigested) in comparison to the amount of substrate that entered the gel (product) reported as a percent digested.
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    The coordinates were then translated such that the centre of mass was closer to the origin, (0,0,0), and prepared for docking with AutoDockTools.
    AutoDockTools
    suggested: None
    Blind docking was carried out using AutoDock Vina using a grid box of 64 Å x 70 Å x 126 Å centred on (-0.476, 5.300, 8.298), encompassing the entire protein surface.
    AutoDock
    suggested: (AutoDock, RRID:SCR_012746)
    Docked poses were analysed using PyMOL and rendered with ChimeraX.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    ChimeraX
    suggested: (UCSF ChimeraX, RRID:SCR_015872)
    Flash column chromatography was performed using a Biotage Isolera automated flash column chromatography platform using Biotage KP-SNAP-Sil or SNAP Ultra columns.
    SNAP
    suggested: (SNAP, RRID:SCR_007936)
    The data were plotted and IC50 values obtained in Graphpad Prism v8.3.0.
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.08.13.248211v1 on bioRxiv