Protein-primed RNA synthesis in SARS-CoVs and structural basis for inhibition by AT-527
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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.
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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 Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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-nsp8suggested: (Acris Antibodies GmbH Cat# AP09089SU-N, RRID:AB_2035808)anti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Resources SARS-CoV cofactor protein nsp10 was expressed under the control of a Tet-promoter in a pASK vector in E. coli NEB Express … 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 Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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-nsp8suggested: (Acris Antibodies GmbH Cat# AP09089SU-N, RRID:AB_2035808)anti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Resources 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. C2523suggested: NoneSARS-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). C2566suggested: NoneSoftware and Algorithms Sentences Resources 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 Dakosuggested: NoneAll movies were automatically recorded using SerialEM (Mastronarde, 2005) at a magnification of 105K, with a physical pixel size of 0.83 Å. SerialEMsuggested: (SerialEM, RRID:SCR_017293)CTF estimation, 2D classification, 3D classification and refinements were all performed in cryoSPARC. cryoSPARCsuggested: (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). Cootsuggested: (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.
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