Detection of NPC1 binding to viral proteins on virions

To purify NPC1 proteins, HEK293T cells were cultured in 13 10-cm dishes and transfected with 9 µg pcDNA3.1-NPC1-3xFLAG per dish using PEI. After 48 h, cells were lysed with 1 mL pre-cold buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5% glycerol, 1% DDM) with anti-protease inhibitor cocktail per dish at 4 °C for 30–40 min. The whole cell lysate was further centrifuged at 13,000 × g for 10 min at 4 °C to get rid of insoluble fractions. Supernatants were mixed with anti-FLAG beads (Sigma) and rotated overnight at 4 °C. These NPC1-captured beads were collected and washed 5 times with PBS. HIV-1 pseudovirions were produced from HEK293T cells by transfection with pNL-∆Env-Luc and a vector expressing EBOV-GP or SARS2-S. Virions were purified by ultracentrifugation at 100,000 × g for 3 h and resuspended in PBS. Purified virions were digested with thermolysin for 1 h at 37 °C and digestion was terminated by adding 10 mM Phosporamidon. Digested virions were incubated with NPC1-captured beads for 1 h at room temperature. Unbound virions were removed by washing beads with PBS 4-5 times. Bead-associated proteins were detached by adding the lysis buffer along with 1x SDS-PAGE loading buffer and analyzed by Western blotting.