Proteoforms of the SARS-CoV-2 nucleocapsid protein are primed to proliferate the virus and attenuate the antibody response

  1. Corinne A. Lutomski
  2. Tarick J. El-Baba
  3. Jani R. Bolla
  4. Carol V. Robinson

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Abstract

The SARS-CoV-2 nucleocapsid (N) protein is the most immunogenic of the structural proteins and plays essential roles in several stages of the virus lifecycle. It is comprised of two major structural domains: the RNA binding domain, which interacts with viral and host RNA, and the oligomerization domain which assembles to form the viral core. Here, we investigate the assembly state and RNA binding properties of the full-length nucleocapsid protein using native mass spectrometry. We find that dimers, and not monomers, of full-length N protein bind RNA, implying that dimers are the functional unit of ribonucleoprotein assembly. In addition, we find that N protein binds RNA with a preference for GGG motifs which are known to form short stem loop structures. Unexpectedly, we found that N undergoes proteolytic processing within the linker region, separating the two major domains. This process results in the formation of at least five proteoforms that we sequenced using electron transfer dissociation, higher-energy collision induced dissociation and corroborated by peptide mapping. The cleavage sites identified are in highly conserved regions leading us to consider the potential roles of the resulting proteoforms. We found that monomers of N-terminal proteoforms bind RNA with the same preference for GGG motifs and that the oligomeric state of a C-terminal proteoform (N 156-419 ) is sensitive to pH. We then tested interactions of the proteoforms with the immunophilin cyclophilin A, a key component in coronavirus replication. We found that N 1-209 and N 1-273 bind directly to cyclophilin A, an interaction that is abolished by the approved immunosuppressant drug cyclosporin A. In addition, we found the C-terminal proteoform N 156-419 generated the highest antibody response in convalescent plasma from patients >6 months from initial COVID-19 diagnosis when compared to the other proteoforms. Overall, the different interactions of N proteoforms with RNA, cyclophilin A, and human antibodies have implications for viral proliferation and vaccine development.

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  1. Version published to 10.1101/2020.10.06.328112v2 on bioRxiv
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    SciScore for 10.1101/2020.10.06.328112: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Ethics: Patients were recruited from the John Radcliffe Hospital in Oxford, United Kingdom, between March and May 2020 with written and informed consent.
    IRB: Ethical approval was given by the South Central–Oxford C Research Ethics Committee in England (reference: 13/SC/0149), Scotland A Research Ethics Committee (reference: 20/SS/0028) and World Health Organization Ethics Review Committee (RPC571 and RPC572l; 25 April 2013).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Western Blot: Recombinant antibody generated from the full-length SARS-CoV-2 nucleocapsid protein (product ab272852) and anti-human secondary antibody were purchased from Abcam.
    SARS-CoV-2 nucleocapsid protein
    suggested: (Bioss Cat# bsm-41414M, RRID:AB_2848129)
    anti-human secondary antibody were purchased from Abcam.
    suggested: None
    Enzyme-Linked Immunosorbent Assay: Recombinant antibody generated from the full-length SARS-CoV-2 nucleocapsid protein (product ab272852) and goat anti-human IgG (ab97225) conjugated with horseradish peroxidase (HRP) purchased from Abcam.
    anti-human IgG
    suggested: (Abcam Cat# ab97225, RRID:AB_10680850)
    HRP
    suggested: None
    Software and Algorithms
    SentencesResources
    Monoisotopic masses of fragment ions generated by HCD having a normalized intensity of 10% or higher were fed into Prosite Lite software.
    Prosite
    suggested: (PROSITE, RRID:SCR_003457)
    LC-MS data were searched against both the E. coli proteome manually annotated with the SARS-CoV-2 nucleocapsid protein sequence using MaxQuant v1.6.17.0.
    MaxQuant
    suggested: (MaxQuant, RRID:SCR_014485)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    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 10.1101/2020.10.06.328112v1 on bioRxiv