Architecture and self‐assembly of the SARS‐CoV ‐2 nucleocapsid protein

  1. Qiaozhen Ye
  2. Alan M. V. West
  3. Steve Silletti
  4. Kevin D. Corbett

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Abstract

The COVID‐2019 pandemic is the most severe acute public health threat of the twenty‐first century. To properly address this crisis with both robust testing and novel treatments, we require a deep understanding of the life cycle of the causative agent, the SARS‐CoV‐2 coronavirus. Here, we examine the architecture and self‐assembly properties of the SARS‐CoV‐2 nucleocapsid protein, which packages viral RNA into new virions. We determined a 1.4 Å resolution crystal structure of this protein's N2b domain, revealing a compact, intertwined dimer similar to that of related coronaviruses including SARS‐CoV. While the N2b domain forms a dimer in solution, addition of the C‐terminal spacer B/N3 domain mediates formation of a homotetramer. Using hydrogen‐deuterium exchange mass spectrometry, we find evidence that at least part of this putatively disordered domain is structured, potentially forming an α‐helix that self‐associates and cooperates with the N2b domain to mediate tetramer formation. Finally, we map the locations of amino acid substitutions in the N protein from over 38,000 SARS‐CoV‐2 genome sequences. We find that these substitutions are strongly clustered in the protein's N2a linker domain, and that substitutions within the N1b and N2b domains cluster away from their functional RNA binding and dimerization interfaces. Overall, this work reveals the architecture and self‐assembly properties of a key protein in the SARS‐CoV‐2 life cycle, with implications for both drug design and antibody‐based testing.

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  1. Version published to 10.1002/pro.3909
  2. ScreenIT

    SciScore for 10.1101/2020.05.17.100685: (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.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    10006625; NCBI RefSeq YP_009724397) and inserted by ligation-independent cloning into UC Berkeley Macrolab vector 2B-T (AmpR, N-terminal His6-fusion; Addgene #29666) for expression in E. coli.
    RefSeq
    suggested: (RefSeq, RRID:SCR_003496)
    Peptides were identified using PLGS version 2.5 (Waters, Inc.), deuterium uptake was calculated using DynamX version 2.0 (Waters Corp.), and uptake was corrected for back-exchange using DECA 51.
    PLGS
    suggested: None
    Uptake plots were generated in Prism version 8.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    Diffraction datasets were processed with the RAPD pipeline (https://github.com/RAPD/RAPD/), which uses XDS 52 for indexing and data reduction, and the CCP4 programs AIMLESS 53 and TRUNCATE 54 for scaling and conversion to structure factors.
    CCP4
    suggested: (CCP4, RRID:SCR_007255)
    The resulting model was manually rebuilt in COOT 56 and refined in phenix.
    COOT
    suggested: (Coot, RRID:SCR_014222)
    All structural figures were generated with PyMOL version 2.3.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)

    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.

    About SciScore

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  3. Version published to 10.1101/2020.05.17.100685v2 on bioRxiv
  4. Version published to 10.1101/2020.05.17.100685v1 on bioRxiv