Nucleocapsid mutations in SARS-CoV-2 augment replication and pathogenesis

  1. Bryan A. Johnson
  2. Yiyang Zhou
  3. Kumari G. Lokugamage
  4. Michelle N. Vu
  5. Nathen Bopp
  6. Patricia A. Crocquet-Valdes
  7. Birte Kalveram
  8. Craig Schindewolf
  9. Yang Liu
  10. Dionna Scharton
  11. Jessica A. Plante
  12. Xuping Xie
  13. Patricia Aguilar
  14. Scott C. Weaver
  15. Pei-Yong Shi
  16. David H. Walker
  17. Andrew L. Routh
  18. Kenneth S. Plante
  19. Vineet D. Menachery

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Abstract

While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203–205 in the SARS-CoV-2 nucleocapsid protein. Recreating a mutation found in the alpha and omicron variants in an early pandemic (WA-1) background, we find that the R203K+G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. The R203K+G204R mutant corresponds with increased viral RNA and protein both in vitro and in vivo . Importantly, the R203K+G204R mutation increases nucleocapsid phosphorylation and confers resistance to inhibition of the GSK-3 kinase, providing a molecular basis for increased virus replication. Notably, analogous alanine substitutions at positions 203+204 also increase SARS-CoV-2 replication and augment phosphorylation, suggesting that infection is enhanced through ablation of the ancestral ‘RG’ motif. Overall, these results demonstrate that variant mutations outside spike are key components in SARS-CoV-2’s continued adaptation to human infection.

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  1. Version published to 10.1371/journal.ppat.1010627
  2. Version published to 10.1101/2021.10.14.464390v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.10.14.464390: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    All raw sequencing data will be available in the NCBI Small Read Archive with BioProject.
    NCBI Small Read Archive
    suggested: None
    BioProject
    suggested: (NCBI BioProject, RRID:SCR_004801)

    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.

    Results from scite Reference Check: We found no unreliable references.

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  4. Version published to 10.1101/2021.10.14.464390v1 on bioRxiv