293T cells seeded on 6-well plates were cotransfected with 0.8 μg HIV-1–NL4.3–inGluc vector plus 0.4 μg SARS-CoV-2 spike-coding plasmid. Forty-eight hours after transfection, viral supernatant was harvested and used to infect target cells. Unless otherwise indicated, 293T/ACE2 cells were used for infection. (A) Schematic representation of the pseudoviral production and infection. Note that Gluc activity can only be detected in virus-infected target cells — and not in the virus-producing cells — because of the presence of an intron inserted in the sense of the vector that splits the Gluc gene into 2 parts. (B and C) Titers of HIV-1 inGluc pseudotypes bearing the spikes of SARS-CoV (n = 6), SARS-CoV-2 (n = 6), or VSV-G (n = 3); absolute luciferase readouts at 48 hours after infection, and relative infectivity compared with the background, were plotted, respectively. (D) Indicated doses of viral supernatant were used to infect 293T/ACE2 cells seeded in 24-well plates, and 20 μL of supernatant of virus-infected cells were used to measure the Gluc activity as shown. The dashed line indicates the background of luciferase activity; n = 3. (E) Indicated amounts of culture media harvested from virus-infected cells were used to measure Gluc activity; n = 3. (F) Relative infectivity of HIV-inGluc pseudotypes bearing S proteins of SARS-CoV, SARS-CoV-2, or VSV-G in indicated target cells, with parental or those overexpressing ACE2; n = 6. Data were analyzed as mean ± SD.