SARS-CoV-2 nucleocapsid protein drives pulmonary injury by enhancing GRP75-dependent ER-mitochondria tethering and reprogramming of alveolar macrophages.

AIMS: To determine whether the SARS-CoV-2 nucleocapsid (N) protein promotes lung injury by augmenting GRP75-dependent endoplasmic reticulum (ER)-mitochondria tethering and reprogramming alveolar macrophages (AMϕs). MATERIALS AND METHODS: MAMs, ER, and mitochondria were isolated from mouse lungs. Bone marrow-derived and RAW264.7 macrophages were stimulated with recombinant N protein ± lentiviral GRP75 knockdown. ER-mitochondria interactions, tethering complex formation, mitochondrial Ca2+ dynamics, and function were assessed by confocal imaging, transmission electron microscopy, proximity ligation assay, co-immunoprecipitation, and biochemical approaches. In vivo, GRP75 was knocked down in C57BL/6 mouse lungs prior to intratracheal N protein challenge. Lung injury, AMϕ polarization, mitochondrial function, and single-cell transcriptomic changes were assessed. KEY FINDINGS: N protein promoted redistribution of GRP75 to MAMs without altering total lung GRP75 levels. It enhanced ER-mitochondria contacts and IP3R1-GRP75-VDAC1 complex assembly, resulting in excessive mitochondrial Ca2+ accumulation, membrane depolarization, increased mitochondrial reactive oxygen species, ATP depletion, and proinflammatory cytokine production. GRP75 knockdown reversed these defects, restoring mitochondrial function and suppressing inflammation. In vivo, pulmonary GRP75 knockdown alleviated N protein-induced lung injury, inflammatory cell infiltration, cytokine release, M1 polarization, and mitochondrial dysfunction in AMϕs. Single-cell RNA sequencing further demonstrated that GRP75 knockdown reversed N protein-induced AMϕ transcriptional reprogramming by reducing the proinflammatory Car4+ subset while expanding an anti-inflammatory CD74+ subset. SIGNIFICANCE: These findings establish GRP75-dependent ER-mitochondria tethering as a critical mechanism by which N protein disrupts AMϕ homeostasis and promotes COVID-19-associated lung injury, identifying GRP75-mediated MAM signaling as a potential therapeutic target.