Dissecting Interactions between RNA and Coronavirus Nucleocapsid Proteins Using Native Mass Spectrometry

Area of Science:

• Virology
• Structural Biology
• Biochemistry

Background:

• The SARS-CoV nucleocapsid protein (N protein) is crucial for viral RNA packaging and storage.
• Disordered regions in the N protein complicate understanding its RNA interactions.
• Sequence variations across SARS-CoV variants may modulate these interactions.

Purpose of the Study:

• To investigate protein-RNA and protein-protein interactions of SARS-CoV-1 WT, SARS-CoV-2 WT, and SARS-CoV-1 omicron nucleocapsid proteins.
• To understand how sequence variations influence these interactions.
• To identify RNA binding sites and assess complex stabilities.

Main Methods:

• Native mass spectrometry to analyze protein complexes and RNA binding.
• Ultraviolet photodissociation (UVPD) to localize RNA binding sites.
• Energy-variable collision-induced dissociation and variable temperature electrospray ionization to assess complex stability and interaction strengths.

Main Results:

• All three N proteins exist as monomers and dimers, binding up to two RNA molecules per monomer, indicating dual RNA binding sites.
• RNA binding sites were localized to the RNA binding domain and N-terminus.
• SARS-CoV-1 N protein dimers showed lower stability in the gas phase compared to SARS-CoV-2 and omicron variants.
• Protein-RNA interactions are stronger than protein-protein interactions in solution.

Conclusions:

• Nucleocapsid protein interactions with RNA are complex, involving dual binding sites and varying stabilities across SARS-CoV variants.
• Protein-RNA interactions dominate over protein-protein interactions in solution.
• Understanding these interactions is key to deciphering viral RNA packaging and potential therapeutic targets.