FIG 5.

Benztropine and cyproheptadine inhibit pseudotyped Marburg virus at the late-entry stage. (a) Pseudotyped Marburg virus was incubated with A549 cells at 4°C at the −1 h time point. After 1 h of incubation, the virus was removed and temperature was shifted to 37°C to trigger virus internalization. Heparin (10 μg/ml), cyproheptadine (25 μM), benztropine (25 μM), bafilomycin A1 (100 nM), or AZT (1 μM) was introduced at different time points of virus infection, and the drugs' effects on viral infection are shown. (b) Pseudotyped Marburg virus containing Vpr-GFP was incubated with A549 cell in the presence of DMSO (control), benztropine (25 μM), cyproheptadine (25 μM), or heparin (10 μg/ml) at 4°C for 15 min. Virus was then removed, and temperature was shifted to 37°C to trigger virus internalization in the continued presence of drugs or control. After 2 h of incubation, the cells were washed with cold PBS, fixed, stained for nuclei using DAPI, and analyzed by microscopy. Virions inside A549 cells were indicated by green fluorescent foci. (c) Quantification of pseudotyped Marburg virions inside A549 cells. Virion uptake was quantified by counting all the green fluorescent foci inside cells in each image (8 to 14 cells/image). A total of 55 to 68 cells were analyzed for each drug treatment. The data are presented as the average numbers of virions per cell with standard deviations.