FIG. 2.

Compound 7 binds to EBOV-GP and inhibits virus infection. (A) Schematic diagram of a “single-cycle time-of-addition experiment” with HIV/EBOV-GP to determine the stage of virus entry blocked by compound 7. This experiment was designed to characterize the mechanism of action of the antiviral compounds. (B) A single-cycle time-of-addition experiment was done with HIV/EBOV-GP to determine the stage of EBOV entry blocked by compound 7. 293T cells were infected with 100 μl of p24-normalized HIV/EBOV-GP. Compound 7 was added and left for 1 h before infection (−1 h), for 1 h during adsorption (0 h), and for 1 h after infection (+1 h). Infected monolayers were washed with PBS and incubated for 72 h. Inhibition of HIV/EBOV-GP pseudotype infection was detected as a reduced luciferase signal. Error bars indicate standard deviations. (C) A Microcon Ultracell YM3 centrifugal device (Millipore) was used to study the binding of compound 7 to HIV/EBOV-GP and HIV/VSV-G pseudotype viruses. Pseudotype viruses (or a no-virus buffer control) were incubated with 25 μM compound 7 at room temperature for 60 min, and the virus-compound mixture was then loaded onto the Microcon Ultracell YM3 centrifugal device. After centrifugation, the flowthrough was analyzed for unbound compound by high-pressure liquid chromatography. The percentage of bound compound was calculated based on the peak area compared to that of a no-virus control. Error bars indicate standard deviations.