Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex

  1. James Chen
  2. Brandon Malone
  3. Eliza Llewellyn
  4. Michael Grasso
  5. Patrick M. M. Shelton
  6. Paul Dominic B. Olinares
  7. Kashyap Maruthi
  8. Ed Eng
  9. Hasan Vatandaslar
  10. Brian T. Chait
  11. Tarun Kapoor
  12. Seth A. Darst
  13. Elizabeth A. Campbell

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Abstract

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp8 2 /nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryo-electron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template-product in complex with two molecules of the nsp13 helicase. The Nidovirus-order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12-thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg 2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapeutic development.

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    SciScore for 10.1101/2020.07.08.194084: (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
    Nsp12. SARS-CoV-2 RNA was derived from the supernatant of propagated Vero E6 cells and provided by B.R. tenOever (Blanco-Melo et al., 2020).
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Protein cloning: All constructs were verified by sequencing (GeneWiz).
    GeneWiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    For data processing, the acquired MS spectra were visualized using Thermo Xcalibur Qual Browser (v. 4.2.47)
    Thermo Xcalibur Qual Browser
    suggested: None
    Dose-fractionated movies were gain-normalized, drift-corrected, summed, and dose-weighted using MotionCor2 (Zheng et al., 2017).
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    The contrast transfer function (CTF) was estimated for each summed image using the Patch CTF module in cryoSPARC v2.15.0 (Punjani et al., 2017).
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Models were inspected and modified in Coot (Emsley and Cowtan, 2004).
    Coot
    suggested: (Coot, RRID:SCR_014222)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 33, 35, 37, 39 and 12. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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 10.1101/2020.07.08.194084 on bioRxiv