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).
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