Kinetics of CXCR4 and CCR5 tropic Virus-Cell Fusion. (A) Magi +/+ cells were plated at 10,000 cells/well in two separate 96 well plates. Cells were infected with wild type HIV-1NL4.3 or HIV-1JRFL and incubated at 4°C for 2 hours to allow viral binding. Unbound virus was washed away with PBS. Both plates were incubated for an additional 2 hours; one plate remaining at 4°C, while the other plate was shifted to 23°C-TAS. Following the second 2-hour incubation period, both plates were shifted directly to 37°C to promote viral fusion. AMD 3100 (4 uM) or TAK 779 (5 ug/mL) were added at various time points to inhibit subsequent fusion. (A, C) Virus-cell fusion kinetics are faster when pre-incubated at 23°C-TAS (diamonds) compared to when cells are directly shifted to 37°C following the 4°C incubation period (squares). (B, D) CXCR4 and CCR5 fusion kinetics after 4 hours. Varying temperatures were maintained using Eppendorf Centrifuge 5810 R. Relative infectivity was quantified using a liquid β-Gal assay. OD 405 nm, optical density at 405 nm. The averages of triplicate experiments are shown (n = 4). Error bars represent the standard error of the mean.