HIV Pseudovirus Entry Assay

CEM.NKR-CCR5 cells (9 × 10⁵ cells in 24-well plates containing RPMI supplemented with 20 μg/mL of Polybrene) were infected with 500 ng of p24 of luciferase-reporter pseudoviruses, bearing VNP- or RP-Envs, in 1 mL total volume with RPMI 1640 for 2 hours (by centrifugation at 1,200 rpm at 25 °C) and subsequent incubation for 4 hours at 37 °C, as previously described^{5 ,21 (link),22 (link)}. As a control for R5-tropic viral infection, a BaL.01-env plasmid (catalog number 11445, NIH AIDS Research and Reference Reagent Program) was used. Unbound virus was then removed by washing the infected cells. After 48 hours of infection, luciferase activity was measured using a luciferase assay kit (Biotium, Hayward, CA) with a microplate reader (VictorTM X5; PerkinElmer, Waltham, MA). When indicated, permissive cells were pretreated with an anti-CD4 neutralizing mAb (5 μg/mL; eBioscience, San Diego, CA).