Luciferase assay kit

HeLa-P5 cells (20,000 cells in 96-well plates with 20 μg/mL of Polybrene) were infected with a synchronous dose of luciferase-based X4- or R5-tropic HIV-1 viral inputs (500 ng of p24), in 200 μl RPMI 1640 medium for 5 h at 37°C. Virus was then removed by washing and subsequent trypsinization of infected cells. After 48 h of infection, luciferase activity (associated to viruses entry into infected cells) was determined from cell lysates by using a luciferase assay kit (Biotium, Hayward, CA, USA) with a microplate reader (VictorTM X5, PerkinElmer, Waltham, MA, USA), as described [80 (link), 83 (link)]. Similarly, HIV-1 infection was measured by measuring β-galactosidase enzymatic activity in 48 h-infected cells (activity associated to cell lysates from infected HeL-P5 cells) using a β-Gal reporter gene assay (Roche Diagnostics), as previously described [44 (link), 83 (link)]. Inhibition of luciferase or β-galactosidase activity was calculated for each dose point after subtracting background (in the presence of a neutralizing anti-CD4 mAb at 5 μg/mL), and the background of luciferase measurement/β-galactosidase activity in non-infected cells. Data were analyzed using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA).

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).

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 Env, and were produced under the different experimental conditions of this study (see previous point for viral production) in 1 mL of total volume with RPMI 1640 for 2 h (by centrifugation at 1200× g at 25 °C), with subsequent incubation for 4 h at 37 °C, as previously described [12 (link),19 (link),20 (link),21 (link),22 (link),23 (link),25 (link),63 (link),64 (link),65 (link),66 (link),67 (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.