Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates

  1. Adriana Savastano
  2. Alain Ibáñez de Opakua
  3. Marija Rankovic
  4. Markus Zweckstetter

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Abstract

The etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription, and is essential for viral replication. Here, we show that RNA induces cooperative liquid–liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid–liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.

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  1. Version published to 10.1038/s41467-020-19843-1
  2. ScreenIT

    SciScore for 10.1101/2020.06.18.160648: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    Materials: Alexa Fluor 594 conjugated anti G3BP1 antibody was purchased from Santa Cruz Biotechnology (sc-365338
    Materials: Alexa Fluor 594 conjugated anti G3BP1 antibody
    suggested: None
    anti G3BP1
    suggested: None
    A mixture of 0.5 µm Alexa Fluor 488 lysine labeled NSARS-CoV-2 and 1:100 Alexa Fluor 594 conjugated G3BP1 antibody (Santa Cruz Biotechnology, sc-365338 AF594) in cell permeabilization buffer was added to the cells and movies were recorded with 512 x 512 pixel resolution at 1000 Hz speed and 1 s per frame for about 2-3 minutes.
    G3BP1
    suggested: (Santa Cruz Biotechnology Cat# sc-365338, RRID:AB_10846950)
    Experimental Models: Cell Lines
    SentencesResources
    The mobile and immobile fractions were calculated using the parameters a and c derived from each fitting, according to the following equations:

    Stress granule co-localization and FRAP: HeLa cells (DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, ACC 57) were grown in an incubator at 37 °C in humidified atmosphere with 5 % CO2.

    HeLa
    suggested: DSMZ Cat# ACC-57, RRID:CVCL_0030)
    Software and Algorithms
    SentencesResources
    PolyU was added to the mixture to reach a final concentration of 1 µM.
    PolyU
    suggested: (Hong Kong Polytechnic University; Hong Kong; China, RRID:SCR_011289)
    DIC and fluorescent micrographs were acquired on a Leica DM6B microscope with a 63× objective (water immersion) and processed using Fiji software (NIH).
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    Spectra were processed with TopSpin 3.6 (Bruker) and analyzed using Sparky (Lee et al., 2015).
    TopSpin
    suggested: (TopSpin, RRID:SCR_014227)
    The MD simulations were carried out in GROMACS (version 2018.3) using the AMBER99SB-ILDN force field and the TIP3P water model at a temperature of 300 K, 1 bar of pressure and with a coupling time (ζT) of 0.1 ps.
    GROMACS
    suggested: (GROMACS, RRID:SCR_014565)
    The number of contacts and secondary structure over the simulation trajectory were analyzed using the PyMOL Molecular Graphics System (Version 1.8.4.0, Schrödinger, LLC).
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 10. 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:
    • 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.

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  3. Version published to 10.1101/2020.06.18.160648v1 on bioRxiv