SARS-CoV-2 ORF3a blocks lysosomal cholesterol egress by disrupting VPS39-regulated NPC2 trafficking and BMP metabolism.

Cholesterol homeostasis depends on lysosomes liberating cholesterol from degraded lipids. We show that SARS-CoV-2 blocks lysosomal cholesterol egress through the viral protein ORF3a. ORF3a binds the HOPS subunit VPS39 via the W193 and Y184 residues. Disrupting this interface restores cholesterol trafficking. Mechanistically, the ORF3a-VPS39 interaction exerts dual effects. First, it traps the retromer complex on endolysosomes, preventing endosome-to-Golgi recycling and mislocalizing the cholesterol transporter NPC2. Retromer deletion reproduced these defects, whereas the ORF3a W193A mutant restored retromer trafficking. Second, ORF3a-VPS39 interaction reduces bis(monoacylglycerol)phosphates (BMPs), lysosomal lipids required for cholesterol egress, by disrupting the transfer of their precursor, phosphatidylglycerols, from mitochondria. Lipidomics revealed increased mitochondrial and decreased lysosomal phosphatidylglycerol metabolites. Disturbing autophagy or mitochondrion-derived vesicles did not alter BMP levels, whereas ORF3a reduced mitochondrion-lysosome membrane contacts. These findings identify dual functions of VPS39-regulating retromer trafficking and BMP biosynthesis-and also reveal how ORF3a blocks lysosomal cholesterol egress.