Distinct Roles of SARS-CoV-2 N Protein and NFP in Host Cell Response Modulation

The SARS-CoV-2 nucleocapsid (N) protein is crucial for viral replication and modulation of host cell responses. Here, we identify and characterize a novel N-fusion protein, designated NFP. NFP is derived from an alternative open reading frame spanning the N gene and the non-structural protein 1 (NSP1) sequence. While NFP shares some functional domains with the canonical N protein, it exhibits distinct structural features and protein interactions. NFP retains the ability to dimerize and bind RNA but lacks the formation of biomolecular condensates associated with N. Notably, NFP can dominantly interfere with N’s condensate formation capacity when co-expressed. Functionally, NFP partially suppresses stress granule (SG) formation through a G3BP1-independent mechanism but gains the ability to interact with G3BP1 in the presence of N, potentially through N-NFP heterodimer formation. Post-translational modifications, particularly ubiquitination of specific lysine residues (K374 in N and K502 in NFP), differentially regulate the subcellular localization, SG inhibition, and cell cycle regulation activities of N and NFP. Our findings establish NFP as a distinct viral effector protein that modulates host cellular environments through both conserved and unique mechanisms compared to the canonical N protein, providing insights into SARS-CoV-2 pathogenesis and potential therapeutic targets.