Production of SARS-CoV-2 Spike protein pseudotyped lentivirus

Pseudotyped lentiviral particles expressing SARS-CoV-2 Spike protein were produced and titered as previously described (^{Crawford et al., 2020}). HEK293T cells were seeded at a density of 5 × 10⁵ cells per well in 6-well plates containing DMEM (supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin/streptomycin/fungizone). After incubating for 16-25 hours, cells were transfected using FuGENE-6 (Promega) with the Luciferase_IRES_ZsGreen backbone, the Gag/Pol-, Rev-, and Tat lentiviral helper plasmids, and a plasmid containing the codon-optimized Spike sequence from the Wuhan-Hu-1 strain. The Spike sequence contained a 21 amino acid deletion at the cytoplasmic tail (also known as HDM_Spikedelta21). After incubating for 24 hours, we replaced media with fresh supplemented DMEM. Between 50-60 hours post-transfection, viral supernatants were collected, filtered through a 0.22 m Steriflip filter and stored at −80°C. To titer pseudovirus, 1.25 × 10⁴ HEK293T cells expressing ACE2 were plated in a volume of 50 uL in 96-well black-walled plates and incubated for 16-24 hours before addition of 100 uL viral supernatant to each well. Pseudovirus supernatants were diluted 1:10 in supplemented DMEM, followed by seven 2-fold serial dilutions. Each dilution was run in duplicate. Sixty hours after infection, 100 uL of media was removed from each well and 30 uL of Bright-Glo reagent (Promega) was added. Relative luciferase units (RLU) were then measured on a LUMIstar Omega plate reader.