Genomic RNA Elements Drive Phase Separation of the SARS-CoV-2 Nucleocapsid

Abstract

We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with viral RNA. N-protein condenses with specific RNA genomic elements under physiological buffer conditions and condensation is enhanced at human body temperatures (33 C and 37 C) and reduced at room temperature (22 C). RNA sequence and structure in specific genomic regions regulate N-protein condensation while other genomic regions promote condensate dissolution, potentially preventing aggregation of the large genome. At low concentrations, N-protein preferentially crosslinks to specific regions characterized by single-stranded RNA flanked by structured elements and these features specify the location, number, and strength of N-protein binding sites (valency). Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is RNA sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules, and therefore presents a screenable process for identifying antiviral compounds effective against SARS-CoV-2.

Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids

“…While phase boundaries are commonly explored varying ligand concentrations, ionic strength, or pH (Cubuk et al, 2020;Perdikari et al, 2020;Savastano et al, 2020), we apply temperature changes as a driving parameter. This complements the recent study by Iserman et al observing temperature-dependent LLPS by light microscopy (Iserman et al, 2020). Our biophysical data suggest that condensates form in two stages.…”

Programmed −1 Ribosomal Frameshifting in coronaviruses: A therapeutic target

“…This cryo-EM reconstruction also includes a mini-helix formed within the slippery site and spacer region upstream of the pseudoknot. However, RNA chemical modification studies of the SARS-CoV-2 genome reveal that the slippery site is single-stranded ( Huston et al, 2020 ; Iserman et al, 2020 ; Lan et al, 2020 ; Manfredonia et al, 2020 ; Zhang et al, 2020 ), suggesting that the mini-helix is an artifact of the construct used for cryo-EM imaging.…”

Structure and regulation of coronavirus genomes: state-of-the-art and novel insights from SARS-CoV-2 studies

“…Multiple computational and experimental analyses provided compelling evidences that the N protein has a high propensity to undergo liquid-liquid phase separation (LLPS), that is stimulated by interaction with the viral gRNA [78][79][80][81]. This interaction preferentially occurs with single-stranded RNA regions flanked by stably structured elements [82], further suggesting that LLPS might be exploited by SARS-CoV-2 for efficient genome packaging. Of the several interacting host cell proteins identified, the vast majority appears to have a host-protective effect, as their knockout resulted into virus-induced cell death [31], and to be part of the RNA-protein interactome of other positive-sense RNA viruses such as Zika virus (ZIKV) and DENV [30,31].…”