ATP and nucleic acids competitively modulate LLPS of the SARS-CoV2 nucleocapsid protein.

ABSTRACT

SARS-CoV-2 nucleocapsid (N) protein with very low mutation rates is the only structural protein which not only functions to package viral genomic RNA, but also manipulates host-cell machineries, thus representing a key target for drug development. Recent discovery of its liquid-liquid phase separation (LLPS) opens up a new direction for developing anti-SARS-CoV-2 strategies/drugs. However, so far the high-resolution mechanism of its LLPS still remains unknown. Here by DIC and NMR characterization, we have demonstrated 1) nucleic acids modulate LLPS by dynamic and multivalent interactions over both folded NTD/CTD and Arg/Lys residues within IDRs; 2) ATP with concentrations > mM in all living cells but absent in viruses not only binds NTD/CTD, but also Arg residues within IDRs with a Kd of 2.8 mM; and 3) ATP dissolves nucleic-acid-induced LLPS by competitively displacing nucleic acid from binding the protein. Our study deciphers that the essential binding of N protein with nucleic acid and its LLPS are targetable by small molecules including ATP, which is emerging as a cellular factor controlling the host-SARS-CoV-2 interaction. Fundamentally, our results imply that the mechanisms of LLPS of IDR-containing proteins mediated by ATP and nucleic acids appear to be highly conserved from human to virus.