Mapping SARS-CoV-2 Nucleocapsid Function with Nanobodies.

The SARS-CoV-2 nucleocapsid (N) protein is essential for viral RNA packaging, replication, and immune modulation. Despite its central role, the mechanistic contributions of its individual domains, the N-terminal domain (NTD), C-terminal domain (CTD), and the intrinsically flexible linker (LINK), remain poorly defined, largely due to the protein's structural complexity. In this study, we developed a panel of twelve alpaca-derived nanobodies (VHHs) targeting the NTD, CTD, and LINK regions of N. Using ELISA and biolayer interferometry, we characterized their binding affinities, and we mapped their epitopes via hydrogen-deuterium exchange-mass spectrometry (HDX-MS) and structural modeling. When expressed intracellularly, these VHHs inhibited SARS-CoV-2 infection. In vitro, they disrupted phase separation of the N protein, a critical step in viral replication. Strikingly, VHHs targeting each domain independently blocked both phase condensation and viral replication, underscoring the functional importance of all three regions. These findings establish domain-specific VHHs as versatile tools for dissecting N biology, with promising therapeutic potential.