Phosphoregulation of Phase Separation by the SARS-CoV-2 N Protein Suggests a Biophysical Basis for its Dual Functions

Abstract

The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription. It is not clear how the N protein mediates such distinct functions. The N protein contains two RNA-binding domains surrounded by regions of intrinsic disorder. Phosphorylation of the central disordered region promotes the protein's transcriptional function, but the underlying mechanism is not known. Here, we show that the N protein of SARS-CoV-2, together with viral RNA, forms biomolecular condensates. Unmodified N protein forms partially ordered gel-like condensates and discrete 15-nm particles based on multivalent RNA-protein and protein-protein interactions. Phosphorylation reduces these interactions, generating a more liquid-like droplet. We propose that distinct oligomeric states support the two functions of the N protein: unmodified protein forms a structured oligomer that is suited for nucleocapsid assembly, and phosphorylated protein forms a liquid-like compartment for viral genome processing.

Keywords: COVID-19; Coronavirus; N protein; SARS-CoV-2; biomolecular condensate; nucleocapsid; phase separation; phosphorylation.

Graphical abstract

Figure 1

SARS-CoV-2 N Protein Forms Biomolecular Condensates in the Presence of RNA (A) Top: schematic of N protein domain architecture. NTE, N-terminal extension; NTD, N-terminal domain; SR, SR region; CTD, C-terminal domain; CTE, C-terminal extension; CBP, CTD basic patch. Bottom: features of amino acid sequence. PLAAC, prion-like amino acid composition (Lancaster et al., 2014); NCPR, net charge per residue. See Figure S1 for sequence. (B) Light microscopy images of N protein condensates after a 30-min incubation at room temperature with the indicated RNA molecules. All images are representative of multiple independent experiments; scale bar, 10 μm. (C) Condensate formation by N protein (10 μM) over a range of 5′-400, TRS-10, and Random-10 RNA concentrations. All images are representative of multiple independent experiments; scale bar, 10 μm. Random-10 RNA is a mixed population of 10-nt random sequences. See Figure S3 for condensate formation at other N protein and 5′-400 RNA concentrations.

Figure 2

Disordered Regions Modulate N Protein Condensate Formation (A) Top: schematics of N protein deletion mutants. Bottom: N protein condensates observed in the presence of 5′-400 RNA or TRS-10 RNA after a 30-min incubation at room temperature. Images are representative of multiple independent experiments. Scale bar, 10 μm. (B) Absorbance at 340 nm was used to quantify the turbidity of N protein mixtures after a 15-min incubation at room temperature with 1 μM 5′-400. Data points indicate mean ± SEM of duplicates; representative of two independent experiments.

Figure 3

Phosphorylation Modulates N Protein Condensate Properties (A) Sequences of the SR regions of SARS-CoV N protein (aa 177–210) and SARS-CoV-2 N protein (aa 176–209). Proposed priming sites (P1 and P2) for GSK-3 are indicated (Wu et al., 2009). P2 (S206 in SARS-CoV-2) is a Cdk consensus site (yellow) where phosphorylation is thought to prime sequential phosphorylation (arrows) of five upstream sites (green) by GSK-3. P1 phosphorylation by an unknown kinase primes phosphorylation at three upstream sites (orange). (B) The indicated N protein variants were incubated for 30 min with Cdk1-cyclin B1 and/or GSK-3 and radiolabeled ATP, and reaction products were analyzed by SDS-PAGE and autoradiography. Radiolabeled N protein is indicated. Asterisk indicates cyclin B1 autophosphorylation. Molecular weight marker shown on the right (kDa). (C) N protein was incubated overnight with unlabeled ATP and Cdk1-cyclin B1 (left lanes) or no kinase (right lanes), desalted, and incubated with or without GSK-3 and radiolabeled ATP. Reaction products were analyzed by SDS-PAGE and autoradiography. Molecular weight marker shown on the right (kDa). (D) 10 μM N protein was incubated 2 h with Cdk1-cyclin B1 and GSK-3 in the presence or absence of ATP, dialyzed into droplet buffer overnight, and mixed with 1 μM 5′-400 RNA. After 30 min, N protein condensates were analyzed by light microscopy. (E) Images of N protein 10D mutant following 30-min incubation with or without 1 μM 5′-400 or 10 μM TRS-10 RNA. (F) Images of 10D mutants with the indicated deletions, incubated with or without 1 μM 5′-400 RNA. (B–F) All results are representative of multiple independent experiments; scale bar, 10 μm.

Figure 4

Phosphorylation of N Protein Promotes Liquid-like Behavior (A) N protein (10 μM wild-type [WT] or 10D) was mixed with 1 μM 5′-400 RNA for 20 min, and images were taken at 30-s intervals. Arrows indicate droplet fusion events in the 10D mutant. No fusion events were observed in WT structures. (B) 20 μM N protein was phosphorylated with Cdk1-cyclin B1 and GSK-3 as in Figure 3D, incubated with 1 μM 5′-400 RNA for 20 min, and imaged every 60 s. Representative droplet fusion events are boxed with higher magnification images in insets at upper right. Images are representative of multiple independent experiments; scale bar, 10 μm (2 μm for insets). (C) FRAP analysis of droplets formed with 20 μM dye-labeled N protein (WT or 10D) and 1 μM 5′-400 RNA. Following 30 s of bleaching, droplet fluorescence was measured starting at time zero. Data points indicate mean fluorescence intensity as a percentage of pre-bleaching intensity (mean ± SEM, n = 2 for WT and n = 3 for 10D). Note the break in the y axis to allow better viewing of recovery data. Results are representative of two independent experiments. See Figure S4C for images. (D) 10 μM N protein (WT or 10D) was incubated without or with 1 μM PS-318 RNA for 15 min prior to analysis by negative-stain electron microscopy. Images are representative of three independent experiments. Scale bar, 100 nm. (E) 2D class averages of particles from the EM analysis of WT N protein and RNA in (D). Particle selection was not possible with the nonuniform structures formed by the 10D mutant.