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

Area of Science:

• Virology
• Biophysics
• Molecular Biology

Background:

• The SARS-CoV-2 nucleocapsid (N) protein is crucial for packaging the viral genome into ribonucleoprotein particles.
• The precise molecular mechanisms governing N-protein's genome packaging and its role in liquid-liquid phase separation (LLPS) remain incompletely understood.

Purpose of the Study:

• To investigate the biophysical interactions between the SARS-CoV-2 N-protein and nucleic acids (NA) of varying lengths.
• To elucidate the structural and energetic changes associated with N-protein/NA complex formation and subsequent phase separation.

Main Methods:

• Utilized biophysical techniques to analyze N-protein interactions with oligonucleotides.
• Examined the size, composition, secondary structure, and energetics of resulting N-protein/NA complexes and phase-separated states.

Main Results:

• Observed the formation of supramolecular clusters preceding droplet growth, indicating a nucleation-growth mechanism for phase separation.
• Short oligonucleotides formed compact N-protein/NA complexes with reduced intrinsic disorder.
• Longer oligonucleotides promoted extensive multi-valent interactions, leading to higher-order oligomers and robust droplet formation.

Conclusions:

• Nucleic acid length is a critical determinant in SARS-CoV-2 N-protein complex assembly and phase separation.
• Phase separation involves significant changes in N-protein secondary structure, potentially facilitating ribonucleoprotein particle formation within condensates.