SARS-CoV-2 initiates infection through its spike (S) glycoprotein, which binds the angiotensin-converting enzyme 2 (ACE2) receptor on host cells. The S protein comprises two subunits: S1, containing the receptor-binding domain (RBD) that recognizes ACE2, and S2, which mediates membrane fusion. Following ACE2 binding, host proteases such as TMPRSS2 or cathepsins cleave the S protein, exposing the S2 fusion peptide and enabling viral–host membrane fusion. Consequently, SARS-CoV-2 enters cells either via a TMPRSS2-dependent cell-surface route or an endocytic route requiring cathepsin activation in acidic endosomes.
Recent genome-wide CRISPR screens identified Niemann-Pick C1 (NPC1) as an essential host factor for SARS-CoV-2 infection. Our compound screen revealed Tubeimosides as potent inhibitors of viral entry that specifically target NPC1. NPC1 is a late-endosomal membrane protein that, with NPC2, transports cholesterol but also serves as an intracellular receptor for filoviruses through its luminal domain C (NPC1-C). We found that the SARS-CoV-2 RBD similarly binds NPC1-C and that NPC1 is required for viral entry.
NPC1 knockout cells showed reduced viral entry, particularly in TMPRSS2-negative cells, confirming NPC1’s function in the endocytic, not the cholesterol-transport, pathway. NPC1 loss did not affect SARS-CoV-2–mediated cell–cell or virion–virion fusion, indicating its specific involvement in endosomal fusion. The Omicron variant, which favors endocytic entry, exhibited heightened dependence on NPC1. Live-cell imaging showed spike–NPC1 complexes localized to late endosomes/lysosomes, while biochemical assays revealed enhanced spike–NPC1 binding and diminished spike–ACE2 binding under acidic conditions. Artificial exposure of NPC1-C on the cell surface permitted spike interaction and fusion, further amplified at low pH.
Together, these findings demonstrate that NPC1-C directly mediates membrane fusion in late endosomes. SARS-CoV-2 thus undergoes a receptor switch—from ACE2 to NPC1—during endocytic entry.
