Charge neutralization drives nucleic acid-induced phase separation of the hCoV-HKU1 nucleocapsid protein N-terminal domain.

The coronavirus nucleocapsid protein (N) is an RNA-binding protein essential for nucleocapsid assembly and discontinuous transcription. It comprises two folded domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), linked by an intrinsically disordered region containing a serine-arginine (SR)-rich motif. Here, we investigated the liquid-liquid phase separation (LLPS) ability of a protein construct encompassing the NTD and SR-motif (N-NTD-SR) of the endemic hCoV-HKU1 in vitro. Unlike SARS-CoV-2 N-NTD-SR, hCoV-HKU1 N-NTD-SR formed condensates in the presence of nucleic acid alone, indicating higher LLPS propensity. Heterotypic phase separation was evaluated using specific Transcriptional Regulatory Sequences (ssTRS(+), ssTRS(-), and dsTRS) and nonspecific (ssNS(+), ssNS(-), and dsNS) DNA oligonucleotides. All oligonucleotides triggered N-NTD-SR condensation, albeit at different molar ratios. The resulting condensates were sensitive to NaCl but resistant to 1,6-hexanediol, highlighting the importance of electrostatic interactions. N-NTD-SR:DNA condensates exhibited slow fusion rates, suggesting high viscoelasticity, with fluorescence recovery after photobleaching (FRAP) confirming a semi-liquid state. Using protein mutants, where solvent-exposed aromatic residues in hCoV-HKU1 N-NTD were replaced with SARS-CoV-2 equivalents, and chimeras, where the hCoV-HKU1 N-NTD was fused to the SARS-CoV-2 SR-motif, we found that the SR-motif drives phase separation, while the NTD regulates solubility. Furthermore, hCoV-HKU1 N-NTD-SR underwent homotypic condensation at physiological temperature, exhibiting lower critical solution temperature behavior. These findings shed light on the molecular mechanisms governing hCoV-HKU1 N-NTD-SR phase separation and provide a foundation for antiviral strategies aimed at its inhibition.