Colorimetric reverse transcriptase assay

SARS-CoV-2 Spike pseudotyped lentivirus was produced as previously described (Crawford et al., 2020 ). Briefly, HEK293T cells were transfected with 1 μg pHAGE-CMV-Luc2-IRES-ZsGreen-W (BEI), a lentiviral backbone plasmid expressing luciferase under a CMV promoter and an IRES followed by ZsGreen, 0.22 μg HDM-Hgpm2 (BEI), a lentiviral helper plasmid expressing HIV Gag-Pol under a CMV promoter, 0.22 μg HDM-tat1b (BEI), a lentiviral helper plasmid expressing HIV Tat under a CMV promoter, 0.22 μg pRC-CMV-Rev1b (BEI), a lentiviral helper plasmid expressing HIV Rev under a CMV promoter, and 0.34 μg of the plasmid encoding HDM-SARS2-Spike-delta21 using polyethylenimine (Polyplus) in serum-free Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich) supplemented with 25 mM HEPES buffer (Corning). Media was changed to D10 24h post-transfection. After 48h, pseudotyped lentivirus was harvested by filtering supernatant through a 0.45 μm low protein binding durapore membrane (Millipore). Frozen aliquots were stored at −80°C and viral concentrations were quantified using the colorimetric Reverse Transcriptase Assay (Sigma-Aldrich). All packaging plasmids were propagated in DH5α cells (NEB).