Virus Infections

HEK-293T and Caco-2 (ATCC) cells were grown in DMEM media supplemented with 10% fetal calf serum (FCS), pen/strep, L-Glutamine, and passaged 1:8 every three days. For the transfection of NRP1, ACE2 and TMPRSS1, 8 mm glass coverslips were coated with 0.01% poly-L-lysine for 1 hour at 37°C and 5×105 cells were plated in each well of 24-well plates. Cells

were also plated on 96-well imaging plates (PerkinElmer) at a dilution of 1×104 cells per well in 100 μl of full growth medium. 24 hours after plating, cells were transfected in Opti-MEM supplemented with GlutaMAX (Gibco) and 5% FCS with Lipofectamine 3000 reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, 0.25 μg (24-well plates) of DNA per well was prepared in Opti-MEM and P3000 reagent (for Lipofectamine 3000). The DNA was then mixed with the transfection reagent and left at room temperature for 5 minutes. Cells were then incubated with the mix for 4 hours for Lipofectamine)  before the transfection reagent was replaced with fresh growth media. The viral particles were added 24 hours after transfection. After treatment, the cells were fixed in 4% paraformaldehyde (PFA) for 20 min.