HIV-1 Pseudovirus Infection Assay

CEM.NKR-CCR5 cells (9 × 10⁵ cells in 24-well plates with 20 μg/mL of polybrene) were infected with 200 ng of p24 of luciferase-reporter pseudoviruses, bearing R5-tropic BaL (BaL.01-env plasmid, catalog number 11445, NIH AIDS Research and Reference Reagent Program), X4-tropic HXB2 (env, catalogue number 5040154, NIH AIDS Research and Reference Reagent Program) or primary Envs in 1 mL total volume with RPMI 1640 for 2 h (by centrifugation at 1200× g at 25 °C) and subsequent incubation for 4 h at 37 °C, as previously described [24 (link),25 (link),26 (link),28 (link),29 (link),45 (link),65 (link)]. Unbound viruses were then removed by washing the infected cells. After 24 h of infection, luciferase activity was measured using a luciferase assay kit (Biotium, Hayward, CA, USA) with a microplate reader (VictorTM X5; PerkinElmer, Waltham, MA, USA). Anti-CD4 neutralizing mAb L3T4 (5 μg/mL) was used as a control for the blockade of HIV-1 infection by preincubating permissive CEM.NKR-CCR5 cells with this mAb for 30 min at 37 °C before adding viral input.