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.