To generate (HIV/NanoLuc) SARS-CoV-2 pseudotype particles, 5 × 106 293T cells were plated per 10-cm dish in 10 ml in growth medium. The following day, 7.5 µg pHIV-1NL4-3 ΔEnv-NanoLuc reporter virus plasmid and 2.5 µg SARS-CoV-2 or SARS-CoV plasmid (unless otherwise indicated, pSARS-CoV-2-SΔ19 was used) were mixed mix thoroughly with 500 µl serum-free DMEM (this represents a molar plasmid ratio of 1:0.55). Then, 44 µl polyethylenimine (PEI; 1 mg/ml) was diluted in 500 µl serum-free DMEM and mixed thoroughly.
To generate control virus lacking S, the S expression plasmid was omitted from the transfection, and the amount of PEI was reduced to 30 µl. The diluted DNA and PEI were then mixed thoroughly by pipetting or vortexing, incubated at 20 min at room temperature (RT), and added dropwise to the 293T cells. After 8-h or overnight incubation, the transfected cells were washed carefully twice with PBS and incubated in 10 ml DMEM++. At 48 h after transfection, the 10-ml supernatant was harvested, clarified by centrifugation at 300 g for 5 min, and passed through a 0.22-µm pore-size polyvinylidene fluoride syringe filter (Millipore; SLGVR33RS), aliquoted, and frozen at −80°C.
To generate control virus lacking S, the S expression plasmid was omitted from the transfection, and the amount of PEI was reduced to 30 µl. The diluted DNA and PEI were then mixed thoroughly by pipetting or vortexing, incubated at 20 min at room temperature (RT), and added dropwise to the 293T cells. After 8-h or overnight incubation, the transfected cells were washed carefully twice with PBS and incubated in 10 ml DMEM++. At 48 h after transfection, the 10-ml supernatant was harvested, clarified by centrifugation at 300 g for 5 min, and passed through a 0.22-µm pore-size polyvinylidene fluoride syringe filter (Millipore; SLGVR33RS), aliquoted, and frozen at −80°C.
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