Protein-primed RNA synthesis in SARS-CoVs and structural basis for inhibition by AT-527

  1. Ashleigh Shannon
  2. Véronique Fattorini
  3. Bhawna Sama
  4. Barbara Selisko
  5. Mikael Feracci
  6. Camille Falcou
  7. Pierre Gauffre
  8. Priscila El Kazzi
  9. Etienne Decroly
  10. Nadia Rabah
  11. Karine Alvarez
  12. Cécilia Eydoux
  13. Jean-Claude Guillemot
  14. Françoise Debart
  15. Jean-Jacques Vasseur
  16. Mathieu Noel
  17. Adel Moussa
  18. Steven Good
  19. Kai Lin
  20. Jean-Pierre Sommadossi
  21. Yingxiao Zhu
  22. Xiaodong Yan
  23. Hui Shi
  24. François Ferron
  25. Bruno Canard

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

How viruses from the Coronaviridae family initiate viral RNA synthesis is unknown. Here we show that the SARS-CoV-1 and −2 Ni dovirus R dRp- A ssociated N ucleotidyltransferase (NiRAN) domain on nsp12 uridylates the viral cofactor nsp8, forming a UMP-Nsp8 covalent intermediate that subsequently primes RNA synthesis from a poly(A) template; a protein-priming mechanism reminiscent of Picornaviridae enzymes. In parallel, the RdRp active site of nsp12 synthesizes a pppGpU primer, which primes (-)ssRNA synthesis at the precise genome-poly(A) junction. The guanosine analogue 5’-triphosphate AT-9010 (prodrug: AT-527) tightly binds to the NiRAN and inhibits both nsp8-labeling and the initiation of RNA synthesis. A 2.98 Å resolution Cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-(nsp8) 2 /RNA/NTP quaternary complex shows AT-9010 simultaneously binds to both NiRAN and RdRp active site of nsp12, blocking their respective activities. AT-527 is currently in phase II clinical trials, and is a potent inhibitor of SARS-CoV-1 and −2, representing a promising drug for COVID-19 treatment.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2021.03.23.436564: (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

    Antibodies
    SentencesResources
    Nsp8 was probed with mouse monoclonal anti-nsp8 (5A10) from GeneTex (GTX632696), and HRP-conjugated rabbit anti-mouse secondary (Agilent Dako), washing 3X in PBS.T between each antibody.
    anti-nsp8
    suggested: (Acris Antibodies GmbH Cat# AP09089SU-N, RRID:AB_2035808)
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV cofactor protein nsp10 was expressed under the control of a Tet-promoter in a pASK vector in E. coli NEB Express C2523 cells (New England Biolabs) carrying the pRare2LacI (Novagen) plasmid.
    C2523
    suggested: None
    SARS-CoV nsp14 was expressed from a pDEST14 vector in E. coli strain NEB Express C2566 cells (New England Biolabs) carrying the pRare2, in the presence of Ampicillin (100 μM/mL) and Chloramphenicol (17 μg/mL).
    C2566
    suggested: None
    Software and Algorithms
    SentencesResources
    Nsp8 was probed with mouse monoclonal anti-nsp8 (5A10) from GeneTex (GTX632696), and HRP-conjugated rabbit anti-mouse secondary (Agilent Dako), washing 3X in PBS.T between each antibody.
    Agilent Dako
    suggested: None
    All movies were automatically recorded using SerialEM (Mastronarde, 2005) at a magnification of 105K, with a physical pixel size of 0.83 Å.
    SerialEM
    suggested: (SerialEM, RRID:SCR_017293)
    CTF estimation, 2D classification, 3D classification and refinements were all performed in cryoSPARC.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    The model was manually built in Coot (Emsley et al., 2010) with the guidance of the Cryo-EM map, and in combination with real space refinement using Phenix (Afonine et al., 2018).
    Coot
    suggested: (Coot, RRID:SCR_014222)

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 33. 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 10.1101/2021.03.23.436564v1 on bioRxiv