Characterisation of the SARS-CoV-2 ExoN (nsp14 ExoN -nsp10) complex: implications for its role in viral genome stability and inhibitor identification
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Abstract
The SARS-CoV-2 coronavirus (CoV) causes COVID-19, a current global pandemic. SARS-CoV-2 belongs to an order of Nidovirales with very large RNA genomes. It is proposed that the fidelity of CoV genome replication is aided by an RNA nuclease complex, formed of non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Here, we confirm that the SARS-CoV-2 nsp14-nsp10 complex is an RNase. Detailed functional characterisation reveals nsp14-nsp10 is a highly versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3’-terminus. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading and purges the nascent replicating RNA strand of a range of potentially replication terminating aberrations. Using the developed assays, we identify a series of drug and drug-like molecules that potently inhibit nsp14-nsp10, including the known Sars-Cov-2 major protease (M pro ) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for bifunctional inhibitors in the treatment of COVID-19.
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SciScore for 10.1101/2020.08.13.248211: (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
Experimental Models: Cell Lines Sentences Resources The protein was generated by transformation into E. coli BL21 Rosetta2 cells and expression in Terrific Broth media supplemented with 30 μg mL-1 kanamycin and 10 mM ZnCl2. Rosetta2suggested: NoneSoftware and Algorithms Sentences Resources Gel images collected on a Typhoon scanner were analysed in Image J to determine the amount of substrate remaining in the well (undigested) in comparison to the amount of substrate that entered the gel … SciScore for 10.1101/2020.08.13.248211: (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
Experimental Models: Cell Lines Sentences Resources The protein was generated by transformation into E. coli BL21 Rosetta2 cells and expression in Terrific Broth media supplemented with 30 μg mL-1 kanamycin and 10 mM ZnCl2. Rosetta2suggested: NoneSoftware and Algorithms Sentences Resources Gel images collected on a Typhoon scanner were analysed in Image J to determine the amount of substrate remaining in the well (undigested) in comparison to the amount of substrate that entered the gel (product) reported as a percent digested. Image Jsuggested: (ImageJ, RRID:SCR_003070)The coordinates were then translated such that the centre of mass was closer to the origin, (0,0,0), and prepared for docking with AutoDockTools. AutoDockToolssuggested: NoneBlind docking was carried out using AutoDock Vina using a grid box of 64 Å x 70 Å x 126 Å centred on (-0.476, 5.300, 8.298), encompassing the entire protein surface. AutoDocksuggested: (AutoDock, RRID:SCR_012746)Docked poses were analysed using PyMOL and rendered with ChimeraX. PyMOLsuggested: (PyMOL, RRID:SCR_000305)ChimeraXsuggested: (UCSF ChimeraX, RRID:SCR_015872)Flash column chromatography was performed using a Biotage Isolera automated flash column chromatography platform using Biotage KP-SNAP-Sil or SNAP Ultra columns. SNAPsuggested: (SNAP, RRID:SCR_007936)The data were plotted and IC50 values obtained in Graphpad Prism v8.3.0. Graphpad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)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: We did not find any issues relating to colormaps.
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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