The “time-of-addition” experiment was designed as previously described (7, 8, 14) to determine the mechanism of action of the antiviral compounds, and the procedure is shown schematically in fig. S2. Briefly, A549 cells were seeded into 96-well plates at a density of 5 × 103 cells per well 24 hours before infection and incubated at 37°C with 5% CO2. Then, the A549 cells were infected with HIV/EBOV GP and treated with test compounds (0.5 μM Y11, 2 μM Y18, or 2 μM E-64d) at the various stages of viral entry. Duplicate wells were used for each time point, and control-infected cultures were treated with only a drug vehicle (DMSO). At 72 hours after infection, the cells were assayed, and the infectivity was measured as described above.

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