The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins

  1. Kristina V. Tugaeva
  2. Dorothy E. D. P. Hawkins
  3. Jake L. R. Smith
  4. Oliver W. Bayfield
  5. De-Sheng Ker
  6. Andrey A. Sysoev
  7. Oleg I. Klychnikov
  8. Alfred A. Antson
  9. Nikolai N. Sluchanko

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Abstract

The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent K D to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association can regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, while hijacking cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.

Highlights

SARS-CoV-2 nucleocapsid protein (N) binds to all seven human 14-3-3 isoforms. This association with 14-3-3 strictly depends on phosphorylation of N. The two proteins interact in 2:2 stoichiometry and with the Kd in a μM range. Affinity of interaction depends on the specific 14-3-3 isoform. Conserved Ser197-phosphopeptide of N is critical for the interaction.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.26.424450: (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
    Protein expression and purification: The tagless SARS-CoV-2 N gene coding for Uniprot ID P0DTC9 protein sequence was commercially synthesized (GeneWiz) and cloned into pET-SUMO bacterial expression vectors using NdeI and HindIII restriction endonuclease sites.
    GeneWiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    Protein concentration was determined by spectrophotometry at 280 nm on a N80 Nanophotometer (Implen, Munich, Germany) using sequence-specific extinction coefficients calculated using the ProtParam tool in ExPASy (see Supplementary table 5).
    ProtParam
    suggested: (ProtParam Tool, RRID:SCR_018087)
    ExPASy
    suggested: None
    Graphing and fitting were performed in Origin 9.0 (OriginLab Corporation, Northampton, MA, USA)
    OriginLab Corporation
    suggested: (Origin, RRID:SCR_014212)
    All processing was performed in ASTRA 8.0 software (Wyatt Technologies) taking dn/dc equal to 0.185 and using extinction coefficients listed in Supplementary table 5.
    ASTRA
    suggested: (ASTRA, RRID:SCR_016255)

    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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  2. Version published to 10.1101/2020.12.26.424450 on bioRxiv