Cryo-EM Structures of the SARS-CoV-2 Endoribonuclease Nsp15

  1. Monica C. Pillon
  2. Meredith N. Frazier
  3. Lucas B. Dillard
  4. Jason G. Williams
  5. Seda Kocaman
  6. Juno M. Krahn
  7. Lalith Perera
  8. Cassandra K. Hayne
  9. Jacob Gordon
  10. Zachary D. Stewart
  11. Mack Sobhany
  12. Leesa J. Deterding
  13. Allen L. Hsu
  14. Venkata P. Dandey
  15. Mario J. Borgnia
  16. Robin E. Stanley

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Abstract

New therapeutics are urgently needed to inhibit SARS-CoV-2, the virus responsible for the on-going Covid-19 pandemic. Nsp15, a uridine-specific endoribonuclease found in all coronaviruses, processes viral RNA to evade detection by RNA-activated host defense systems, making it a promising drug target. Previous work with SARS-CoV-1 established that Nsp15 is active as a hexamer, yet how Nsp15 recognizes and processes viral RNA remains unknown. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15. The UTP-bound cryo-EM reconstruction at 3.36 Å resolution provides molecular details into how critical residues within the Nsp15 active site recognize uridine and facilitate catalysis of the phosphodiester bond, whereas the apo-states reveal active site conformational heterogeneity. We further demonstrate the specificity and mechanism of nuclease activity by analyzing Nsp15 products using mass spectrometry. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.11.244863: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    All plasmids were verified by DNA sequencing (GeneWiz).
    GeneWiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    Beam-induced motion and drift were corrected using MotionCor2 (28) and aligned doseweighted images were used to calculate CTF parameters using CTFFIND4 (29).
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    CryoSPARC v2 (30) was used in all subsequent image processing.
    CryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Fit was improved using a combination of rigid body and real-space refinement in Phenix (31) combined with iterative rounds of building in COOT (32)
    COOT
    suggested: (Coot, RRID:SCR_014222)
    Molprobity (33) was used to evaluate the model and the model statistics are listed in Table 1.
    Molprobity
    suggested: (MolProbity, RRID:SCR_014226)
    Figures and videos were prepared with PyMOL, Chimera, and ChimeraX (34-36).
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    bioRxiv doi: https://doi.org/10.1101/2020.05.20.064436, (2020). 11. 12. Protein Sci 29, 1596-1605 (2020). J. S. Joseph et al.
    bioRxiv
    suggested: (bioRxiv, RRID:SCR_003933)

    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 Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap used on page 19. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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. ScreenIT

    SciScore for 10.1101/2020.08.11.244863: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    All plasmids were verified by DNA sequencing (GeneWiz).
    GeneWiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    Beam-induced motion and drift were corrected using MotionCor2 (28) and aligned dose-weighted images were used to calculate CTF parameters using CTFFIND4 (29).
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    CryoSPARC v2 (30) was used in all subsequent image processing.
    CryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Fit was improved using a combination of rigid body and real-space refinement in Phenix (31) combined with iterative rounds of building in COOT (32)
    COOT
    suggested: (Coot, RRID:SCR_014222)
    Molprobity (33) was used to evaluate the model and the model statistics are listed in Table 1.
    Molprobity
    suggested: (MolProbity, RRID:SCR_014226)
    Figures and videos were prepared with PyMOL, Chimera, and ChimeraX (34–36).
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 19. 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.

  3. Version published to 10.1101/2020.08.11.244863v1 on bioRxiv