SARS-CoV-2 nucleocapsid protein engages with viral RNA and ERGIC lipids to drive viral core assembly

Severe acute respiratory syndrome coronavirus 2 assembles at the ER–Golgi intermediate compartment (ERGIC), yet the molecular basis of nucleocapsid (N) protein interactions with host membranes remains unclear. Using in vitro reconstituted lipid membranes and viral RNA– N complexes, we show that full-length N binds phosphatidylinositol (PI)- and phosphatidylserine (PS)-containing membranes and induces lipid clustering, an effect amplified by viral RNA and ERGIC-like membrane lipid composition. The isolated N-terminal domain lacks this activity, whereas the C-terminal domain retains membrane-associated multimerization. Although, lipid bilayers promote co-clustering of N and PI lipids, facilitating ribonucleoprotein (RNP) assembly, even on simple membranes. Importantly, ERGIC- mimicking membranes enhances this co-clustering further stabilizing RNPs of dimensions matching the viral core. In cells, viral RNA enhances N clustering without altering particle production. These findings reveal cooperative interactions between N, viral RNA, and ERGIC lipids as key drivers of lipid-dependent viral core formation, providing a mechanistic framework for the early steps of viral assembly.