A specific phosphorylation-dependent conformational switch in SARS-CoV-2 nucleocapsid protein inhibits RNA binding

Area of Science:

• Virology
• Structural Biology
• Molecular Mechanisms

Background:

• The nucleocapsid protein (N) of SARS-CoV-2 is vital for encapsidating the viral genome.
• Its serine-arginine-rich (SR) domain undergoes hyperphosphorylation in infected cells, regulating function.
• The precise mechanisms by which hyperphosphorylation affects N protein function remain unclear.

Purpose of the Study:

• To elucidate the structural consequences of SARS-CoV-2 N protein hyperphosphorylation.
• To investigate how specific host kinases modulate N protein-RNA interactions.
• To understand the role of phosphorylation in viral genome packaging and unpackaging.

Main Methods:

• Nuclear magnetic resonance (NMR) spectroscopy was employed to monitor structural changes.
• In vitro phosphorylation assays were performed using specific kinases: serine arginine protein kinase 1 (SRPK1), glycogen synthase kinase 3 (GSK3), and casein kinase 1 (CK1).
• RNA binding assays were conducted to assess the impact of phosphorylation on N protein-RNA interactions.

Main Results:

• Hyperphosphorylation of eight specific sites on the SR domain by SRPK1, GSK3, and CK1 abrogates RNA binding.
• Phosphorylation induces structural changes, causing the SR domain to bind the same interface as single-stranded RNA.
• Phosphorylation by protein kinase A (PKA) did not inhibit RNA binding, highlighting kinase-specific regulatory patterns.
• Long-range contacts involving RNA binding, linker, and dimerization domains were disrupted.

Conclusions:

• Specific patterns of N protein hyperphosphorylation by host kinases directly inhibit RNA binding.
• This inhibition is linked to structural rearrangements affecting key functional domains.
• The findings offer insights into kinase recruitment for regulating SARS-CoV-2 replication and genome management.