A pH-driven receptor switch from ACE2 to NPC1 enables endosomal entry of SARS-CoV-2

Abstract SARS-CoV-2 enters cells either by TMPRSS2-mediated fusion at the cell surface or by cathepsin-dependent fusion from late endosomes, yet how spike remains competent for fusion during endocytic trafficking is unclear. Here we identify Niemann–Pick C1 (NPC1) as an essential intracellular receptor that enables spike-mediated fusion in acidic late endosomes/lysosomes. Genetic ablation or pharmacological inhibition of NPC1 selectively blocked endosomal entry but not the surface entry, and Omicron showed heightened NPC1 dependence consistent with reduced fusogenicity and preferential endosomal entry in cell culture. A bimolecular fluorescence complementation assay localized spike–NPC1 complexes to Rab7/LAMP1-positive compartments. Biochemical and competition analyses, supported by structural modelling, revealed that NPC1 loop C engages a surface on the receptor-binding domain distinct from the ACE2 interface and binds with higher affinity under acidic conditions, whereas ACE2 binding is favored at neutral pH. These findings define a pH-driven receptor switch from ACE2 to NPC1 during endocytic entry.