The Unexpected Visibility of the SARS-CoV-2 Nucleocapsid Protein Reveals a Hidden Route of Surface Trafficking.

The SARS-CoV-2 Nucleocapsid (N) protein, long regarded as an internal structural component of the virion, unexpectedly localizes to the plasma membrane of infected cells. Here, we show that N is actively trafficked to the cell surface via a ceramide-dependent unconventional secretory pathway. Live-stained imaging and kinetic analyses revealed that N surface association begins early in infection, before Spike (S) expression and viral release, and persists after enzymatic removal of heparan sulfate. Pharmacological disruption of phosphoinositide or phosphatidylserine interactions had minimal effect, whereas inhibition of neutral sphingomyelinase with GW4869 markedly reduced surface N, identifying a ceramide-regulated route as essential for its export. This mechanism distinguishes N from canonical transmembrane viral proteins and explains how N-specific antibodies mediate potent Fc-effector responses across SARS-CoV-2 variants. Our findings redefine the spatiotemporal dynamics of coronavirus structural proteins and reveal an unanticipated axis of immune visibility within the infected cell. IMPORTANCEInternal viral proteins are generally thought to remain confined to intracellular compartments, yet several viruses display such proteins at the surface of infected cells through mechanisms that remain incompletely defined. In this study, we characterize a host-regulated trafficking route that contributes to the delivery of the SARS-CoV-2 nucleocapsid (N) protein, a non-membrane viral protein, to the plasma membrane. Our findings indicate that N protein surface expression occurs independently of virion assembly, membrane integration, or extracellular rebinding, and instead involves host vesicular processes outside the classical secretory pathway. By elucidating how a leaderless coronavirus protein can access the cell surface, this work addresses a key gap in coronavirus cell biology and provides a framework for understanding how internal viral proteins may exploit host trafficking pathways. More broadly, these results highlight unconventional host trafficking pathways as determinants of viral protein localization and provide a framework for understanding how viruses exploit cellular export mechanisms beyond the classical secretory system.