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

Adriana Savastano,Marija Rankovic,Markus Zweckstetter,Alain Ibáñez De Opakua

doi:10.1038/s41467-020-19843-1

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.

Highlights

Mean intensity where the fluorescence mean intensity is the averaged droplet intensity calculated by defining its area and the background mean intensity corresponds to the averaged intensity of the background
To investigate if the N protein of SARS-CoV-2 phase separates in the absence of other viral proteins, we measured the turbidity of NSARS-CoV-2 solutions at different protein concentrations
Nucleocapsid assembly can occur outside of liquid-like compartments, it was shown that the rate of assembly is increased when the nucleocapsid protein of Measles virus is concentrated through LLPS15

Summary

Introduction

Mean intensity where the fluorescence mean intensity is the averaged droplet intensity calculated by defining its area and the background mean intensity corresponds to the averaged intensity of the background. For quantification of the partition coefficient of fluorescently labeled RNA, nsp[12], or the RdRp complex, two independent samples were used and a total amount of 100 droplets per condition were analyzed. A twotailed t-test was used to compare the partition coefficient values obtained for either fluorescently labeled RNA, nsp[12], or the RdRp complex recruited into NSARS-CoV-2 (used as a control group) and phosphorylated NSARS-CoV-2. Dynamics of NSARS-CoV-2 molecules in the phase-separated state were investigated by FRAP analysis. NSARS-CoV-2 phase separation was induced using 50 μM NSARSCoV-2 and 1 μM polyU in 20 mM sodium phosphate buffer (NaPi), pH 7.5. For comparison of the kinetics of unmodified and SRPK1 phosphorylated NSARS-CoV-2 protein, droplets of 30 μM nucleocapsid protein induced by the addition of 0.4 μM polyU were used.

Methods

Results

Conclusion