The coronavirus proofreading exoribonuclease mediates extensive viral recombination
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Abstract
Coronaviruses (CoVs) emerge as zoonoses and cause severe disease in humans, demonstrated by the SARS-CoV-2 (COVID-19) pandemic. RNA recombination is required during normal CoV replication for subgenomic mRNA (sgmRNA) synthesis and generates defective viral genomes (DVGs) of unknown function. However, the determinants and patterns of CoV recombination are unknown. Here, we show that divergent β-CoVs SARS-CoV-2, MERS-CoV, and murine hepatitis virus (MHV) perform extensive RNA recombination in culture, generating similar patterns of recombination junctions and diverse populations of DVGs and sgmRNAs. We demonstrate that the CoV proofreading nonstructural protein (nsp14) 3’-to-5’ exoribonuclease (nsp14-ExoN) is required for normal CoV recombination and that its genetic inactivation causes significantly decreased frequency and altered patterns of recombination in both infected cells and released virions. Thus, nsp14-ExoN is a key determinant of both high fidelity CoV replication and recombination, and thereby represents a highly-conserved and vulnerable target for virus inhibition and attenuation.
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SciScore for 10.1101/2020.04.23.057786: (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 MERS-CoV infection: Three nearly confluent 25-cm2 flasks of Vero CCL-81 cells were infected with MERS-CoV at an MOI of 0.3 pfu/cell. Vero CCL-81suggested: NoneSARS-CoV-2 infection: Five subconfluent 25-cm2 flasks of Vero E6 cells were infected at an MOI = 0.45 pfu/cell and cellular monolayers were harvested 60 hpi when the monolayer was <90% fused. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Direct RNA Nanopore … SciScore for 10.1101/2020.04.23.057786: (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 MERS-CoV infection: Three nearly confluent 25-cm2 flasks of Vero CCL-81 cells were infected with MERS-CoV at an MOI of 0.3 pfu/cell. Vero CCL-81suggested: NoneSARS-CoV-2 infection: Five subconfluent 25-cm2 flasks of Vero E6 cells were infected at an MOI = 0.45 pfu/cell and cellular monolayers were harvested 60 hpi when the monolayer was <90% fused. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Direct RNA Nanopore sequencing: RNA from ultracentrifuge-purified virions was prepared for direct RNA Nanopore sequencing on the Oxford Nanopore Technologies MinION platform according to the manufacturer’s protocols. Oxford Nanoporesuggested: (Oxford Nanopore Technologies, RRID:SCR_003756)Libraries were sequenced on fresh MinION R9.4 flow-cells for 24 hours, or until the pore occupancy was under 20%. MinIONsuggested: (MinION, RRID:SCR_017985)Results from OddPub: Thank you for sharing your code and 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 42 and 44. 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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