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

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.

Fig. 1. RNA-induced LLPS of the nucleocapsid protein of SARS-CoV-2.

a Organization of N^SARS-CoV-2 into two globular domains (RNA-binding domain and C-terminal dimerization domain) surrounded by long intrinsically disordered regions (IDR)^,. The serine/arginine (SR)-rich region is conserved in coronaviruses. b Influence of RNA and protein concentration on N^SARS-CoV-2/polyU-LLPS in 20 mM NaPi, pH 7.5, monitored by solution turbidity at 350 nm. Average values from three independent measurements are shown. The dashed line marks N^SARS-CoV-2/polyU-concentrations at which charge neutralization occurs, assuming a charge of −1 per phosphate group. c Fluorescence and DIC microscopy of spherical droplets of 50 µM N^SARS-CoV-2 and 1 µM polyU in 20 mM NaPi, pH 7.5. Fluorescently labeled RNA (green) partitioned into the droplets. Scale bar, 20 µm. Micrographs are representative of three independent biological replicates. d Increase in N^SARS-CoV-2- and RNA-concentration inside of N^SARS-CoV-2/polyU droplets in 20 mM NaPi, pH 7.5. Scale bars 3 µm. Micrographs are representative of three independent biological replicates. e Time-dependent change in diffusion of N^SARS-CoV-2 inside polyU-induced droplets observed by FRAP. FRAP of freshly prepared droplets is shown in green, and after incubation for one hour in blue. Error bars represent the standard deviation for averaged six curves for each time point. Representative micrographs of a fresh droplet (top) and after incubation for one hour (bottom) before bleaching, after bleaching, and at the end of recovery are displayed to the right. Scale bars 10 µm.

Fig. 2. Nucleocapsid protein of SARS-CoV-2 associates with stress granules.

a Alexa Fluor 488 labeled N^SARS-CoV-2 (green) colocalizes with the stress granule marker G3BP1 (red) in arsenite-treated digitonin-permeabilized HeLa cells. Micrographs are representative of three independent biological replicates. b Fluorescence recovery after photobleaching (FRAP) curve fitted with bi-exponential fit suggests the existence of a fast diffusing population of molecules, which together with a slowly recovering population comprises ~65% of the bleached spot (mobile fraction). The remaining 35% are immobile and do not recover its fluorescence after photobleaching. The curve is the average of n = 6 stress granules, error bars represent standard deviation. c Corresponding confocal microscopy pictures of the representative FRAP of N^SARS-CoV-2 associated with stress granules in HeLa cells. Insert shows the bleached stress granule marked with an arrow. Micrographs are representative of n = 6 FRAP experiments in one biological sample. Scale bar 10 µm in (a) and (c), 2 µm in the inset in (c).

Fig. 3. Atomic details of the RNA-interaction of the mutation-prone SR-region.

a Frequency of mutations in the nucleocapsid protein in 42176 SARS-CoV-2 sequences from the China National Center for Bioinformation. Residues with more than 0.0025 frequency are labeled. Domain organization of N^SARS-CoV-2 on top. b NMR-based analysis of the structure of the high-density SR-stretch (residues A182-S197) of N^SARS-CoV-2. Secondary structure derived from chemical shifts using TALOS+ is represented together with the S² order parameter. Arginines are highlighted in gray. c Comparison between the α-helical propensity derived from NMR data (blue) and MD simulations (red). One conformer with α-helical content from the simulation is shown inside the graph. d Comparison between the number of peptide-polyU contacts per peptide in the MD simulations (red; average and standard deviation of five simulations), the NMR chemical shift perturbation (CSP) of the peptide-polyU titration at 1500 nM of polyU (blue), and the frequency of mutations per residue (gray bars). At the left, a MD snapshot of five SR-peptides with the 20 base length polyU is shown.

Fig. 4. SR-phosphorylation modulates RNA-induced condensation of the nucleocapsid protein.

a MD simulations of SR/polyU-interaction. The number of contacts per peptide with a 20 base length polyU is shown in the top graph for the non-phosphorylated and fully phosphorylated SR-peptide as the average and standard deviation of five independent simulations. Snapshots of both peptides are shown. b ¹H 1D experiments of the SR-peptide (1766.8 Da), SPRK1 single-phosphorylated peptide (SR(1 P); 1846.9 Da according to mass spectrometry) and SPRK1 double-phosphorylated peptide (SR(2 P); 1926.9 Da according to mass spectrometry; Supplementary Fig. 8) at three different concentrations of polyU (0, 300 and 1500 nM). The spectral regions, in which the signals of polyU, the backbone H^Ns of unmodified residues and phosphorylated serines are located, are marked. The positive charges of the SR-peptide are compensated by the polyU negative charges at around 300 nM. c Turbidity at 350 nm of solutions of non-phosphorylated (blue) and SRPK1-phosphorylated (red) N^SARS-CoV-2 in 20 mM NaPi, pH 7.5, at 30 µM protein concentration and increasing concentrations of polyU. Average values from three independent measurements are shown. Error bars, std. An SDS-Page gel (insert) displays a band shift due to SRPK1-phosphorylation. d Decreased RNA recruitment into polyU-induced droplets of N^SARS-CoV-2 upon phosphorylation with SRPK1 kinase. Mean values and standard deviation are displayed (n = 100 droplets). Two-sided t test with P value set to < 0.05 for statistical significance, *** < 0.001, ** < 0.002 and * < 0.033, ns < 0.12. e FRAP of N^SARS-CoV-2 (blue) and SRPK1-phosphorylated N^SARS-CoV-2 (red) inside of polyU-induced droplets. Error bars represent the standard deviation for averaged 8 and 10 curves for unmodified and phosphorylated N^SARS-CoV-2, respectively. Representative micrographs of N^SARS-CoV-2 (top) and SRPK1-phosphorylated N^SARS-CoV-2 droplets (bottom) before bleaching, after bleaching and at the end of recovery are displayed to the right. Partition coefficients of 12.2 ± 2.8 and 4.3 ± 1.2 were calculated for N^SARS-CoV-2 and phosphorylated N^SARS-CoV-2, respectively. Scale bars, 10 µm. f Association of unmodified (N^SARS-CoV-2, left panels) and phosphorylated (phosphoN^SARS-CoV-2, right panels) nucleocapsid protein of SARS-CoV-2 with stress granules in HeLa cells, micrographs are representative of two independent biological replicates. Scale bar 20 µm, 5 µm in inset.

Fig. 5. The RNA-dependent RNA polymerase complex of SARS-CoV-2 concentrates in RNA/nucleocapsid protein-droplets.

a Structure of the RNA-bound RdRp-complex formed by the SARS-CoV-2 non-structural proteins nsp12 (green), nsp7 (magenta) and nsp8 (blue) in 1:1:2 stoichiometry (PDB code: 6YYT). b Fluorescence and DIC microscopy show the recruitment of Alexa Fluor 488 labeled nsp12 (green), the catalytic subunit of the RdRp-complex, into N^SARS-CoV-2/polyU droplets (N^SARS-CoV-2 in red). c Active SARS-CoV-2 RdRp-complex bound to a fluorescein-labeled minimal RNA hairpin concentrates inside of N^SARS-CoV-2/polyU droplets. In (b) and (c) droplets of 50 µM N^SARS-CoV-2 and 1 µM polyU were prepared in 20 mM NaPi, pH 7.5, and visualized by the addition of a small amount of Alexa Fluor 594 labeled N^SARS-CoV-2 protein. Scale bars are 10 µm in (b) and (c). Micrographs are representative of three independent biological replicates. d Recruitment of nsp12 and RdRp complex into polyU-induced droplets of unmodified (blue) and SRPK1-phosphorylated (red) N^SARS-CoV-2. Mean values and standard deviation are shown (n = 100 droplets). Two-sided t-test with P value set to < 0.05 for statistical significance, ***<0.001, **<0.002 and *<0.033, ns < 0.12. e FRAP of nsp12 after recruitment into N^SARS-CoV-2/polyU (blue) and SRPK1-phosphorylated N^SARS-CoV-2/polyU (red) droplets. Error bars represent the standard deviation for averaged 10 and 11 curves for unmodified and phosphorylated N^SARS-CoV-2, respectively. (f) Schematic representation of the LLPS-based formation of N/RNA/RdRp-condensates as protein/RNA-dense sites for viral transcription.