FIG. 5.

pH dependence of entry for wild-type, H426A, and H428A EnvA. DF-1 cells were pretreated with 100 nM bafilomycin, chilled to 4°C, and incubated with HIV pseudoparticles containing β-lactamase-Vpr and bearing the indicated EnvA for 1 h at 4°C in the continued presence of 100 nM bafilomycin. The temperature was then raised to 37°C for 20 min, whereupon a predetermined amount of acetic acid was added to adjust the pH to the indicated value. After 6 min at 37°C, the fusion buffer was reneutralized, medium containing bafilomycin was added, and the cells were returned to the incubator for 2 h. The β-lactamase substrate, CCF2A, was then added, and its conversion from a green substrate to a blue product was allowed to proceed at RT overnight. The percentage of cells that were converted from green to blue was determined by flow cytometry. Three independent experiments were performed in duplicate for each virus. The values presented here represent the average of all 6 values at each pH. The error bars represent the standard deviation for each averaged point.