2.6 Production of SARS-CoV-2 S-pseudotyped lentivirus and
pSARS-CoV-2 entry assay
SARS-CoV-2 S-pseudotyped lentiviruses (pSARS-CoV-2) was generated using
2th generation lentiviral packing system as described in previous work
(Kim et al., 2021). Briefly, HEK293T
cells that reached 70-80% confluency were transfected with lentiviral
plasmid harboring a gene encoding for fly luciferase, psPAX2 packing
plasmid, and SARS-CoV-2 S plasmid using Lipofectamine 3000 transfection
reagent (Invitrogen, USA) following the manufacturer’s instructions. At
24 h and 48 h post-transfection, culture supernatants containing
pSARS-CoV-2 virus particles were collected and centrifuged at 500 × g
for 5 min to remove the cellular debris, and stored at 4°C until use.
For pSARS-CoV-2 entry assay, ACE2+ and
ACE2/TMPRSS2+ H1299 cells plated on a 48 well plate
with 60-80% confluency were pre-treated for 1 h with each drugs,
followed by an overlay of supernatants containing pSARS-CoV-2 virus
particles in the presence of each drugs. After 24 h incubation, viral
entry efficiency was quantified by measuring the firefly luciferase
activity in cell lysates using a luciferase assay system (Promega, USA)
and SpectraMax iD5 Multi-Mode Microplate Reader (Molecular Devices,
USA).