SARS-CoV-2 nucleocapsid induces hyperinflammation and vascular leakage through the Toll-like receptor signaling axis in macrophages

Nearly 20% of SARS-CoV-2 infections result in ICU admission, often driven by an imbalance between antiviral responses and inflammatory signaling, leading to uncontrolled cytokine secretion. The SARS-CoV-2 nucleocapsid (N) protein is a known immune antagonist, but its role in macrophage-driven cytokine storms is unclear. We demonstrate that N functions in a stimulus-specific manner, specifically amplifying extracellular and dampening intracellular RNA sensing. Moreover, we show that this is a conserved feature of pathogenic betacoronaviruses through distinct mechanisms. Our interaction networks with SARS-CoV-2 variant N proteins suggest the Delta variant N drives inflammation through interactions with several proteins, most notably, cGAS. Profiling of secreted cytokines revealed that the N proteins disrupt the secretome in a variant-specific manner. Most notably, we found that supernatants from the Delta variant N-expressing macrophages dramatically disrupt heart endothelial barriers, implicating N in COVID-19-associated cardiac complications. Our findings highlight N-mediated immune imbalance as a driver of severe COVID-19 and identify N as a promising therapeutic target to mitigate hyperinflammation.