Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein

  1. Dhurvas Chandrasekaran Dinesh
  2. Dominika Chalupska
  3. Jan Silhan
  4. Vaclav Veverka
  5. Evzen Boura

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the Coronavirus disease 2019 (COVID-19) which is currently negatively affecting the population and disrupting the global economy. SARS-CoV-2 belongs to the +RNA virus family that utilize single-stranded positive-sense RNA molecules as genomes. SARS-CoV-2, like other coronaviruses, has an unusually large genome for a +RNA virus that encodes four structural proteins – the matrix (M), small envelope (E), spike (S) and nucleocapsid phosphoprotein (N) - and sixteen nonstructural proteins (nsp1-16) that together ensure replication of the virus in the host cell. The nucleocapsid phosphoprotein N is essential for linking the viral genome to the viral membrane. Its N-terminal RNA binding domain (N-NTD) captures the RNA genome while the C-terminal domain anchors the ribonucleoprotein complex to the viral membrane via its interaction with the M protein. Here, we characterized the structure of the N-NTD and its interaction with RNA using NMR spectroscopy. We observed a positively charged canyon on the surface of the N-NTD lined with arginine residues suggesting a putative RNA binding site. Next, we performed an NMR titration experiment using an RNA duplex. The observed changes in positions of signals in the N-NTD NMR spectra allowed us to construct a model of the N-NTD in complex with RNA.

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    Table 2: Resources

    Software and Algorithms
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    The 40 structures with the least restraint violations were further refined in explicit solvent using the YASARA software with the YASARA forcefield(13) and subjected to further analysis using the Protein Structure Validation Software suite (www.nesg.org).
    YASARA
    suggested: (YASARA, RRID:SCR_017591)
    The RNA homology model was prepared by mutating the native 7mer RNA duplex (PDB 4U37) (5) in Pymol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.) that was subsequently subjected to an energy minimization in YASARA (13).
    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 11. 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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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.

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  2. Version published to 10.1101/2020.04.02.022194 on bioRxiv