FIG. 2.
(A) Entry and fusion kinetics. The time courses of Ebola virus GP- and VSV-G-pseudotyped virion entry and fusion were studied. Ebola virus GP- or VSV-G-pseudotyped virions were prebound to cells at 4°C for 1 h. After thorough washing to remove unbound viral particles, entry and fusion were initiated by shifting the cells to 37°C. At various times, cells were washed with ice-cold PBS and treated with trypsin to terminate the fusion reaction. Values represent the extent of fusion relative to the maximum level of fusion obtained. (B) Effect of the cholesterol-sequestering agent β-cyclodextrin on Ebola virus GP-, VSV-G-, or HIV-1HXB2 Env-mediated entry and fusion. Cholesterol was depleted with the cholesterol-binding resin β-cyclodextrin. HeLa cells were treated with graded doses of cyclodextrin for 30 min at 37°C, washed three times to deplete the reagent, and incubated with Ebola virus GP-, VSV-G-, or HIV-1HXB2 Env-pseudotyped virions, and entry and fusion were measured. Cells were pretreated with various concentrations of β-cyclodextrin for 30 min at 37°C and thoroughly washed three times with PBS. The cells were then incubated with pseudotyped virions for 3 h at 37°C, and fusion was measured. (C) Effects of bafilomycin A1 on Ebola virus GP-, VSV-G-, and HIV-1HXB2 Env-pseudotyped virus entry and fusion. Cells were pretreated with various concentrations of bafilomycin A1 at 37°C for 1 h and incubated with each of the indicated pseudotyped virions at 37°C for 3 h. Bafilomycin A1 was maintained in the cultures throughout the experiment. Values are levels of fusion relative to that in untreated controls.