The coronavirus proofreading exoribonuclease mediates extensive viral recombination

  1. Jennifer Gribble
  2. Laura J. Stevens
  3. Maria L. Agostini
  4. Jordan Anderson-Daniels
  5. James D. Chappell
  6. Xiaotao Lu
  7. Andrea J. Pruijssers
  8. Andrew L. Routh
  9. Mark R. Denison

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Abstract

Recombination is proposed to be critical for coronavirus (CoV) diversity and emergence of SARS-CoV-2 and other zoonotic CoVs. While RNA recombination is required during normal CoV replication, the mechanisms and determinants of CoV recombination are not known. CoVs encode an RNA proofreading exoribonuclease (nsp14-ExoN) that is distinct from the CoV polymerase and is responsible for high-fidelity RNA synthesis, resistance to nucleoside analogues, immune evasion, and virulence. Here, we demonstrate that CoVs, including SARS-CoV-2, MERS-CoV, and the model CoV murine hepatitis virus (MHV), generate extensive and diverse recombination products during replication in culture. We show that the MHV nsp14-ExoN is required for native recombination, and that inactivation of ExoN results in decreased recombination frequency and altered recombination products. These results add yet another critical function to nsp14-ExoN, highlight the uniqueness of the evolved coronavirus replicase, and further emphasize nsp14-ExoN as a central, completely conserved, and vulnerable target for inhibitors and attenuation of SARS-CoV-2 and future emerging zoonotic CoVs.

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  1. Version published to 10.1371/journal.ppat.1009226
  2. ScreenIT

    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 Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    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-81
    suggested: None
    SARS-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 E6
    suggested: None
    Software and Algorithms
    SentencesResources
    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 Nanopore
    suggested: (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%.
    MinION
    suggested: (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.

    About SciScore

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  3. Version published to 10.1101/2020.04.23.057786v1 on bioRxiv