Luciferase Pseudovirus Infection Assay

CEM.NKR-CCR5 cells (9 × 10⁵ cells in 24-well plates with 20 μg/ml Polybrene) were infected with 500 ng of p24 of luciferase reporter pseudoviruses in 1 ml total volume with RPMI 1640 for 2 h (by centrifugation at 1,200 × g at 25°C) and subsequent incubation for 4 h at 37°C, as previously described (29 (link), 41 (link), 47 (link)). Unbound virus was then removed by washing the infected cells. After 48 h of infection, luciferase activity was measured using a luciferase assay kit (Biotium, Hayward, CA) with a microplate reader (Victor X5; PerkinElmer, Waltham, MA). When indicated, permissive cells were pretreated with an anti-CD4 neutralizing MAb (5 μg/ml; EBioscience, San Diego, CA).