Kinetics of Virus Entry into TZM-bl Cells

To determine virus entry kinetics, TZM-bl cells were seeded in 96-well plates (20.000 cells per well) in complete DMEM supplemented with 10 µg/ml DEAE-Dextran. 24 h post-seeding, cells were first cooled at 4°C for 5 minutes, then the medium was removed. HIV-1 pseudotype virus stocks adjusted to 50.000 RLU in 100 µl DMEM at 4°C were added per well and plates centrifuged for 70 minutes at 1200 g and 10°C. The low temperature was chosen to allow virus attachment during spinoculation but no entry. Following spinoculation the supernatant with unbound virus was removed and 130 µl of DMEM, pre-warmed to 37°C, were added per well to initiate infection (timepoint zero) and plates were incubated at 37°C. At defined timepoints post-infection, 20 µl of T-20 (375 µg/ml in DMEM; yielding a final assay concentration of 50 µg/ml) were added per well to stop the viral entry process. To obtain a measure for infectivity across different experiments, the wells with the last T-20 addition at 120 min after infection start were used as 100% reference infectivity value and the infectivity of all other T-20 treated wells were set in relation to it. In addition, a mock-treated well (addition of 20 µl DMEM at timepoint zero) was evaluated to assess absolute infectivity in absence of T-20.