Despite the odds: formation of the SARS-CoV-2 methylation complex
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Abstract
Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14’s exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.
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SciScore for 10.1101/2022.01.25.477673: (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
Recombinant DNA Sentences Resources Protein expression and complex purification: Constructs of nsp10 comprising amino acids 4254 – 4392, nsp14 comprising amino acids 5926 – 6452, and nsp16 comprising amino acids 6799 – 7096 of SARS-CoV-2 polyprotein 1ab optimized for expression in E. coli were ordered from GeneArt and subcloned into expression vector pETDuet-1. pETDuet-1suggested: RRID:Addgene_170280)Software and Algorithms Sentences Resources The acquired LC-MS/MS data were processed with the use of Proteome Discoverer platform (v.1.4; Thermo Scientific) and searched with an in-house MASCOT server (v.2.5.1; Matrix Science, London, UK) … SciScore for 10.1101/2022.01.25.477673: (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
Recombinant DNA Sentences Resources Protein expression and complex purification: Constructs of nsp10 comprising amino acids 4254 – 4392, nsp14 comprising amino acids 5926 – 6452, and nsp16 comprising amino acids 6799 – 7096 of SARS-CoV-2 polyprotein 1ab optimized for expression in E. coli were ordered from GeneArt and subcloned into expression vector pETDuet-1. pETDuet-1suggested: RRID:Addgene_170280)Software and Algorithms Sentences Resources The acquired LC-MS/MS data were processed with the use of Proteome Discoverer platform (v.1.4; Thermo Scientific) and searched with an in-house MASCOT server (v.2.5.1; Matrix Science, London, UK) against the database of common protein contaminants (cRAP database) with manually added sequences of the proteins of interest. Proteome Discoverersuggested: (Proteome Discoverer, RRID:SCR_014477)Acquired data were converted into Mascot Generic Format (*.mgf) files using built-in scripts and introduced into Mascot search engine (Matrixscience, London, UK), based on SwissProt and in-house created database. Mascotsuggested: (Mascot, RRID:SCR_014322)Image processing and 3-D reconstitution: Collected micrographs were processed using CryoSPARC 3.1.1. CryoSPARCsuggested: (cryoSPARC, RRID:SCR_016501)Initially, 9’350 particles were picked from micrographs using Blob Picker. Blob Pickersuggested: NoneThe nsp10/14/16 complex map derived from SAXS data was used for a rigid-body fit in such 3D-reconstituion map using Dock in map. Docksuggested: (DOCK, RRID:SCR_000128)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 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: 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.
Results from scite Reference Check: We found no unreliable references.
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