Pseudotyping HIV-1 Particles with SARS-CoV-2 Spike

HIV-1 particles pseudotyped with SARS-CoV-2 spike were made as previously described.^{31 (link)} Briefly, a T75 flask was seeded the day before with 3 million HEK293T/17 cells in 10 ml complete Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% foetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin. Cells were transfected using 60 μg of PEI-Max (Polysciences) with a mix of three plasmids: 9.1 μg HIV-1 luciferase reporter vector,^{32 (link)} 9.1 μg HIV p8.91 packaging construct and 1.4 μg wild-type SARS-CoV-2 Spike expression vector.^{32 (link)} Supernatants containing pseudotyped virions were harvested 48 h post-transfection, filtered through a 0.45-μm filter. Infectivity was titrated by serial dilution of supernatant in DMEM (10% FBS and 1% penicillin–streptomycin) followed by addition to HeLa cells (10 000 cells per 100 µl per well) that stably express ACE2 (provided by J. E. Voss, Scripps Institute). After 48–72 h luminescence was assessed as a proxy of infection by lysing cells with the Bright-Glo luciferase kit (Promega), using a Glomax plate reader (Promega).