SARS-CoV-2 Spike Pseudotyped Lentivirus Neutralization

The SARS-CoV-2 spike carrying the D614G mutation pseudotyped lentivirus was produced by transient transfection of HEK293T cells with pLAS2w.EGFP.pPuro, pMDG and pCMV-DR8.91 as previously described [41 (link)]. For the neutralization assay, the nanoparticle preparations were incubated with pseudo virus at 37 °C for 1 h. The mixtures were added to ACE2-expressing H1975 cells (H1975-ACE2), 293T cells (293T-ACE2) or BEAS-2B cells (BEAS-2B-ACE2) and incubated for an additional 4 h. Then, the pseudo virus-containing media were replaced with fresh media. After an additional 72 h, the cells infected with the pseudo virus were detected using an Olympus IX73 Microscope Imaging System (Olympus, Japan). Cells expressing green fluorescent protein (GFP) are considered to be infected with SARS-CoV-2 pseudotyped lentivirus [41 (link)]. For the inhibition studies, H1299 cells were treated with the drug or nanoparticle preparations for 24 h before being infected with virus. After 1 h of infection, the virus-containing media was removed and replaced with fresh media. After incubating for an additional 72 h, the infection efficiency was evaluated by detecting the GFP expression using microscopy. The percent inhibition was calculated by 1-(D/C), in which D and C refer to the presence and absence of quercetin, respectively.