Mechanism of SARS-CoV-2 Nucleocapsid Protein Phosphorylation-Induced Functional Switch

Area of Science:

• Virology
• Molecular Biology
• Structural Biology

Background:

• The SARS-CoV-2 nucleocapsid protein (Np) is crucial for viral RNA replication and packaging.
• Phosphorylation of the Np's Ser-Arg-rich (SRR) linker is hypothesized to regulate these functions.

Purpose of the Study:

• To investigate the mechanistic roles of Np phosphorylation in SARS-CoV-2.
• To elucidate how SRR linker phosphorylation influences Np's RNA binding and complex formation.

Main Methods:

• In vitro biophysical and biochemical assays using recombinant Np.
• Limited proteolysis, circular dichroism spectroscopy, differential scanning fluorimetry.
• Mass photometry (MP) and crosslinking-MP for dimerization and RNA complex analysis.

Main Results:

• Phosphorylation did not significantly alter Np structure or stability but affected dimerization.
• Wild-type Np bound viral RNA stem-loops differently than phosphomimetic variants.
• Binding kinetics and RNA selectivity varied between WT and phosphomimetic Np, with implications for DNA binding.

Conclusions:

• Phosphorylated Np is implicated in RNA replication and chaperoning.
• Non-phosphorylated Np is essential for genomic RNA packaging.
• These findings explain infectivity differences linked to SRR linker variants.