FRET-based virus entry assay

HIV-based pseudovirus incorporating BlaM-Vpr and CoV-2 Spike was produced by transfecting HEK293T cells with pNL4–3E- (15 μg), pCMV4-BlaM-Vpr (5 μg), and pcDNA3.1 CoV-2 Spike (5 μg) using the calcium phosphate technique. Briefly, six million 293T cells were seeded in a T75 flask. Plasmid DNA was mixed with sterile H2O, CaCl2, and Tris-EDTA (TE) buffer, and the totality was combined with Hepes-buffered saline (HBS). The transfection volume was added dropwise, and cells were incubated at 37°C for 48 h. Supernatants were recovered and clarified by centrifugation, passed through a 0.45 μm filter, and stored. Titers were measured using an HIV-1 p24 ELISA kit (XpressBio). 50 ng p25 equivalent of virus was added to HeLa-ACE2 cells for 2 hours. Cells were washed and labeled with the CCF2-AM β-lactamase Loading Kit (Invitrogen) for 2 hours and analyzed for CCF2 cleavage by flow cytometry as described (93). Rapamycin, everolimus, temsirolimus, or ridaforolimus (20 μM) were used to pretreat cells for 4 hours prior to virus addition and were maintained for the duration of infection. DMSO (Sigma) was used as a vehicle control.