Methylation of RNA Cap in SARS-CoV-2 captured by serial crystallography

  1. M. Wilamowski
  2. D.A. Sherrell
  3. G. Minasov
  4. Y. Kim
  5. L. Shuvalova
  6. A. Lavens
  7. R. Chard
  8. N. Maltseva
  9. R. Jedrzejczak
  10. M. Rosas-Lemus
  11. N. Saint
  12. I.T. Foster
  13. K. Michalska
  14. K.J.F. Satchell
  15. A Joachimiak

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

The genome of the SARS-CoV-2 coronavirus contains 29 proteins, of which 15 are nonstructural. Nsp10 and Nsp16 form a complex responsible for the capping of mRNA at the 5′ terminus. In the methylation reaction the S-adenosyl-L-methionine serves as the donor of the methyl group that is transferred to Cap-0 at the first transcribed nucleotide to create Cap-1. The presence of Cap-1 makes viral RNAs mimic the host transcripts and prevents their degradation. To investigate the 2′-O methyltransferase activity of SARS-CoV-2 Nsp10/16, we applied fixed-target serial synchrotron crystallography (SSX) which allows for physiological temperature data collection from thousands of crystals, significantly reducing the x-ray dose while maintaining a biologically relevant temperature. We determined crystal structures of Nsp10/16 that revealed the states before and after the methylation reaction, for the first time illustrating coronavirus Nsp10/16 complexes with the m7 GpppA m2′-O Cap-1, where 2′OH of ribose is methylated. We compare these structures with structures of Nsp10/16 at 297 K and 100 K collected from a single crystal. This data provide important mechanistic insight and can be used to design small molecules that inhibit viral RNA maturation making SARS-CoV-2 sensitive to host innate response.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2020.08.14.251421: (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.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    As new images are acquired, Globus Automate “flows” are launched to process them as follow: 1) moves new files from APS to Theta by using the Globus Transfer service 55; 2) performs DIALS stills_process 56 on batches of 256 images by using funcX 57, a function-as-a-service computation system (funcX uses Parsl 58 to abstract and acquire nodes on Theta as needed, and dispatches tasks to available nodes); 3) extracts metadata from files regarding identified diffractions, and generates visualizations (funcX) showing the locations of positive hits on the mesh; and 4) publishes raw data, metadata, and visualizations to a portal on the ALCF Petrel data system 59.
    Theta
    suggested: (THetA, RRID:SCR_001860)
    Successfully processed images with diffraction-produced integration files were returned to the beamline computers and later refined and merged using PRIME
    PRIME
    suggested: (Prime, RRID:SCR_014887)
    Structure solution and refinement: The crystal structures of the Nsp10/16 complex were solved by molecular replacement using MolRep 61 from the CCP4 package.
    CCP4
    suggested: (CCP4, RRID:SCR_007255)
    The structures were refined by multiple cycles in REFMAC v.
    REFMAC
    suggested: (Refmac, RRID:SCR_014225)
    5.8.0258 62 followed by manual edition of the model using Coot 63.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    The stereochemistry of structures were analyzed using MolProbity and the Ramachandran plot.
    MolProbity
    suggested: (MolProbity, RRID:SCR_014226)
    Figures were prepared with PyMol and UCSF Chimera 64
    PyMol
    suggested: (PyMOL, RRID:SCR_000305)

    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 34, 28 and 29. 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 10.1101/2020.08.14.251421 on bioRxiv