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

## TL;DR

This study explores how phosphorylation of the SARS-CoV-2 nucleocapsid protein changes its function in RNA replication and packaging.

## Contribution

The study reveals how phosphorylation of the nucleocapsid protein's linker region alters its RNA binding and functional roles.

## Key findings

- Phosphorylated nucleocapsid protein preferentially binds to specific viral RNA regions and displays fast binding kinetics.
- Non-phosphorylated nucleocapsid protein compactly binds DNA irreversibly, suggesting a role in RNA packaging.
- Phosphorylation modulates the protein's RNA binding mode from electrostatic to hydrophobic interactions.

## Abstract

The SARS-CoV-2 nucleocapsid protein (Np) is essential for viral RNA replication and genomic RNA packaging. Phosphorylation of Np within its central Ser-Arg-rich (SRR) linker is proposed to modulate these functions. To gain mechanistic insights into these distinct roles, we performed in vitro biophysical and biochemical studies using recombinantly expressed ancestral Np and phosphomimetic SRR variants. Limited-proteolysis showed minor cleavage differences between wild-type (WT) and phosphomimetic Np, but no major structure or stability changes in the N- and C-terminal domains were observed by circular dichroism spectroscopy and differential scanning fluorimetry, respectively. Mass photometry (MP) revealed that WT Np dimerized more readily than phosphomimetic variants. Crosslinking-MP showed that WT Np formed discrete complexes on viral 5′ UTR stem-loop (SL) 5 RNA, whereas phosphomimetic Np assembled preferentially on SL1–4. WT Np bound non-specifically to all RNAs tested primarily via hydrophobic interactions, whereas phosphomimetic Np showed selectivity for SARS-CoV-2-derived RNAs despite binding more electrostatically. A major difference was observed in the binding kinetics; WT Np compacted and irreversibly bound single-stranded DNA, whereas phosphomimetic Np displayed reduced compaction and fast on/off binding kinetics. These mechanistic insights support a model where phosphorylated Np functions in RNA replication and chaperoning, while non-phosphorylated Np facilitates genomic RNA packaging. The findings also help to explain infectivity differences and clinical outcomes associated with SRR linker variants.