The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein

Abstract

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80–90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein’s central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly.

The Integral Role of RNA in Stress Granule Formation and Function

“…Recently, bioinformatic analyses proposed that the N protein of SARS-CoV-2, which contains low complexity domains (LCD) and a high tendency to phase separate, is capable of forming or regulating RNA granules through interactions with host SG-RNAs and proteins ( Cascarina and Ross, 2020 ; Moosa and Banerjee, 2020 ). A series of different studies showed that the N protein does phase separate with RNA and other proteins such as hnRNPs, RNA polymerase, and membrane-associated M protein ( Perdikari et al, 2020 ; Savastano et al, 2020 ; Lu et al, 2021 ). Importantly, phase separation is driven by genomic RNA elements and regulated by phosphorylation of N protein and ATP ( Carlson et al, 2020 ; Iserman et al, 2020 ; Dang et al, 2021 ).…”

Arginine methylation of SARS-Cov-2 nucleocapsid protein regulates RNA binding, its ability to suppress stress granule formation, and viral replication

“…The SG nucleating factor G3BP1 [ 15 , 16 ] and other SG components were identified in the SARS-CoV-2 N protein interactome [ 5 , 17 ], suggesting SARS-CoV-2 like SARS-CoV regulates SGs mainly through N protein. The SARS-CoV-2 N protein is able to form condensates with RNA in vitro [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ], in the cytoplasm of cells [ 21 , 25 ] and partially co-localizes within arsenite-induced SGs [ 26 ]. Several studies have shown that N protein sequesters G3BP1 and disassembles SGs [ 22 , 26 , 27 , 28 ], likely as a means to suppress the host immune response to favor virus replication.…”

“…Several studies have shown that N protein sequesters G3BP1 and disassembles SGs [ 22 , 26 , 27 , 28 ], likely as a means to suppress the host immune response to favor virus replication. Recent studies show that IDR1 and NTD regulate N protein condensates affecting nucleic acid annealing and potentially implicated in viral packaging and assembly [ 21 , 22 , 25 ]. The SR linker region is phosphorylated by SRPK [ 29 ], GSK-3 [ 30 ] and Cdk1-GSK3 [ 18 ], influencing N protein condensates [ 18 , 22 ].…”

Genome-Scale Metabolic Model of Infection with SARS-CoV-2 Mutants Confirms Guanylate Kinase as Robust Potential Antiviral Target

“…[14] observe 38 vRNPs per SARS-CoV-2 virion [14]. Approximately 12 copies of the N protein are located in one vRNP in SARS-CoV-2 [32,33]. Multiplying those two numbers results in 456 copies of the N protein.…”