Hek293t cell

For wildtype PRD-0038 S VSV entry into HEK293T cells transfected with R. affinis, R. sinicus, R. landeri, and R. alcyone alleles full-length ACE2 alleles (Figure 1D), HEK293T cells (ATCC) were cultured in 10% FBS, 1% penicillin–streptomycin DMEM at 37 °C in a humidified 5% CO₂ incubator. Cells were plated 18–24 hours prior to transfection into 96-well plates [3610] (Corning) coated with poly-lysine [P4707] (Sigma). All transfections were performed using full-length Rhinolophus ACE2 placed into a HDM plasmid (synthesized by GenScript). Transfection of ACE2 alleles into HEK293T cells was performed using 0.2 µg DNA and 0.15 µL Lipofectamine 2000 (Life Technologies) per well in Opti-MEM. After a 5 h incubation at 37 °C in a humidified 8% CO₂ incubator, DMEM was added to obtain a final concentration of 10% FBS and 1% penicillin–streptomycin. Cells were incubated at 37 °C in a humidified 8% CO₂ incubator for 36–48 h prior to infection. For each infection test, 2–3 technical replicates were performed, and the assays were repeated on a second day, for a total of 4–6 technical replicates. Three biological replicates (pseudovirus generated on different days) were used for cell entry and each point shown represents the mean fold change for each biological replicate. Results were plotted using Graphpad Prism (Figure 1D). Genbank accession numbers for all ACE2s can be found in Table S1.