Generation of Spike Pseudotyped Viral Particles

To produce seed particles (VSVΔG-luc/GFP + VSV-G), 293T cells were seeded in six-well plates and transfected 24 h later with 2 µg VSV-luc/GFP, 2 µg T7 polymerase, 0.5 µg VSV-N, 0.25 µg VSV-L, 1.25 µg VSV-P and 1 µg VSV-G. VSV seed particles were harvested 48 h post-transfection. Cell supernatants were collected, cleared from cell debris by centrifugation, aliquoted and stored at −80 °C.
CoV spike pseudotypes were produced as previously described^{31 (link)}. 293T cells were seeded onto six-well plates pre-coated with poly-l-lysine (Sigma–Aldrich) and transfected the next day with 1,200 ng of empty plasmid and 400 ng of plasmid encoding coronavirus spike or green fluorescent protein (GFP) as a no-spike control. After 24 h, transfected cells were infected with VSVΔG particles pseudotyped with VSV-G, as previously described^{37 (link)}. After 1 h of incubating at 37 °C, cells were washed three times and incubated in 2 ml DMEM supplemented with 2% FBS, penicillin/streptomycin and l-glutamine for 48 h. Supernatants were collected, centrifuged at 500g for 5 min, aliquoted and stored at −80 °C.