Type I unconventional protein secretion of the SARS-CoV-2 nucleocapsid protein promotes inflammatory cytokine release

SARS-CoV-2 infection remains a global health threat, yet its pathogenic mechanisms are incompletely understood. Here, we show that viral nucleocapsid protein (NP) is detectable in the serum of patients with SARS-CoV-2 infection independently of viral RNA. Using virus-like particle (VLP) systems and in vitro infection models, we demonstrate that NP is secreted in a vesicle-free form via type I unconventional protein secretion (UPS) pathway. This process is regulated by NP phosphorylation and oligomerization, coordinated by viral structural proteins, and dependent on membrane components including heparan sulfate proteoglycans (HSPGs) and phosphatidylinositol 4,5-bisphosphate (PI (4,5) P 2 ). Secreted NP is preferentially taken up by granulocytes and induces the release of inflammatory cytokines, including IL-6 and TNF-α. Our findings shed a light on the secretion mechanism of this pro-inflammatory NP, highlighting it as a potential therapeutic target for COVID-related systemic inflammation.