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.

Characterization of SARS-CoV-2 N protein reveals multiple functional consequences of the C-terminal domain

“…Figure 4A ). N S176D/S188D/S206D displays ~5-fold lower binding affinity to ssRNA compared to N WT binding, a result consistent with previous work examining the impact of LKR phosphorylation on RNA binding(Savastano et al, 2020). We observed a similar trend for the N NTD-LKR construct.…”

“…N WT p1 contains N-RNA with a loose-coil appearance ( Figure 3B, top left ), similar to that observed for other RNA-bound nucleocapsids (Bharat et al, 2012; Mavrakis et al, 2002). Other recent studies have also observed that N protein undergoes liquidliquid phase separation (LLPS) when mixed with RNA(Carlson et al, 2020; Cubuk et al, 2020; Iserman et al, 2020; Jack et al, 2020; Savastano et al, 2020). In agreement with these results, for N WT p2, we mostly observe spheres corresponding to liquid droplets separated from the surrounding buffer ( Figure 3B, top right ).…”

“…Phosphorylation of Ser177 is preceded by phosphorylation of Ser189 and Ser207 (Ser188 and 206 in SARS-CoV-2 N) by other priming kinases (Wu et al, 2009). Moreover, N protein phosphorylation has been qualitatively shown to modulate both RNA binding and phase separation(Carlson et al, 2020; Lu et al, 2020; Savastano et al, 2020). To test if phosphorylation impacts RNA binding and the phase separation of SARS CoV-2 N-RNA, we generated a set of N phosphomimics by mutating specific serine residues to aspartate residues.…”

A novel cell culture system modeling the SARS-CoV-2 life cycle

“…S4A; Table S2), notably, we also found that N protein could interact with G3BP1 and G3BP2, the stress granule assembly proteins, which was in line with previous studies 39, 40 . Recent studies found that N protein could impair the stress granule assembly to escape the antiviral effect 40,41 . Thus, reads, remaining Illumina sequence reads were mapped to human (GRCh38) and SARS2 genome by using HISAT2.1.0 with parameters: --rna-strandness RF -dta.…”

“…Meanwhile, we also identified numerous host factors associated with N protein ( Fig. S4A; Table S2), notably, we also found that N protein could interact with G3BP1 and G3BP2, the stress granule assembly proteins, which was in line with previous studies 39, 40 . Recent studies found that N protein could impair the stress granule assembly to escape the antiviral effect 40,41 .…”