Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures

Abstract

Nucleocapsid protein (N-protein) is required for multiple steps in betacoronaviruses replication. SARS-CoV-2-N-protein condenses with specific viral RNAs at particular temperatures making it a powerful model for deciphering RNA sequence specificity in condensates. We identify two separate and distinct double-stranded, RNA motifs (dsRNA stickers) that promote N-protein condensation. These dsRNA stickers are separately recognized by N-protein's two RNA binding domains (RBDs). RBD1 prefers structured RNA with sequences like the transcription-regulatory sequence (TRS). RBD2 prefers long stretches of dsRNA, independent of sequence. Thus, the two N-protein RBDs interact with distinct dsRNA stickers, and these interactions impart specific droplet physical properties that could support varied viral functions. Specifically, we find that addition of dsRNA lowers the condensation temperature dependent on RBD2 interactions and tunes translational repression. In contrast RBD1 sites are sequences critical for sub-genomic (sg) RNA generation and promote gRNA compression. The density of RBD1 binding motifs in proximity to TRS-L/B sequences is associated with levels of sub-genomic RNA generation. The switch to packaging is likely mediated by RBD1 interactions which generate particles that recapitulate the packaging unit of the virion. Thus, SARS-CoV-2 can achieve biochemical complexity, performing multiple functions in the same cytoplasm, with minimal protein components based on utilizing multiple distinct RNA motifs that control N-protein interactions.

A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2

“…IDRs in the full-length protein are also known to promote liquid-liquid phase separation (LLPS) in the presence of RNA indicating an intricate system of protein-RNA interactions at the basis of the function of the N-protein 12–15 . Both CTD and NTD subdomains can also phase separate from a solution under a variety of external conditions 15,16 . Although the structural details of N-RNA recognition could be of crucial importance for the rational design of novel drugs, current techniques are limited by this Nucleocapsid condensation behaviour in the presence of RNA in solution, which precludes high-resolution structural determination.…”

Nucleoside Analogs and Perylene Derivatives Modulate Phase Separation of SARS-CoV-2 N Protein and Genomic RNA In Vitro

“…First signs of LLPS (7 ± 3 droplets per mm 2 coverslip with a total area equal to 38 ± 10 µm 2 ) appeared at N concentration equal to 3 µM after overnight incubation ( Figure 2 a), and the apparent partitioning coefficient (PC = F droplet /F solution ) was equal to 9 ± 6. For subsequent experiments with RNA, we selected N concentration equal to 3 µM, because it has been used in previous studies of N-gRNA LLPS [ 7 ], is close to the previously reported saturation concentration detected in a cell-free system with non-viral RNA [ 6 ], and can be achieved in infected cells rapidly after virion unpackaging [ 6 , 18 ].…”

“…In particular, they are implicated in replication [ 3 ], immune evasion [ 4 ], and virion packaging [ 5 ]. Justifying the search for specific modulators of the LLPS-mediated steps of the SARS-CoV-2 cycle, the unique molecular grammar of N-gRNA condensates has recently been uncovered [ 6 , 7 ]. It comprises key elements of N and gRNA primary and secondary structures (“stickers”) that define transient heterotypic (N-RNA) or homotypic (N-N) interactions within droplets, as well as droplet plasticity-defining elements (“spacers”) in-between stickers.…”

“…In viral gRNA, two types of stickers have been identified recently. These are dsRNA stretches and YRRRY motifs, where R is a purine nucleotide, and Y is a pyrimidine nucleotide [ 7 , 8 ] ( Figure 1 b).…”

“…The dsRNA stretches are enriched in the ORF1ab-harboring 5′-part of SARS-CoV-2 gRNA and can interact with N RBD2. Such interactions supposedly disrupt N dimers and trigger LLPS by exposing the hydrophobic surface of the DD [ 7 ]. The presumed LLPS-mediated clustering of N at the 5′-terminus of gRNA may repress ORF1ab translation and thus facilitate the generation and translation of subgenomic RNAs (sgRNAs) that encode structural SARS-CoV-2 proteins.…”