FIG 5.

ZCL278 does not affect attachment of Junin virus to the cell surface or endocytosis. (A) Vero cells were incubated with JUNV-A647 for 30 min at 4°C in the presence or absence of 50 μM ZCL278. The A647 fluorescence (Fluo.) intensity associated with the cells, which accounted for bound JUNV, was measured by flow cytometry. Error bars are the means ± SDs from duplicate experiments with at least 10,000 cells per condition. (B) Vero cells were incubated with JUNV at an MOI of 5 for 30 min at 4°C. Cells were extensively washed with cold PBS and lysed for RT-qPCR analysis. The amounts indicated by the histograms are normalized to those for the GAPDH housekeeping gene, and error bars are the means ± SDs from triplicate experiments. (C) Tf-A647 uptake by Vero cells was performed in the presence or absence of 50 μM ZCL278 or 20 μM Dynasore-OH (Dyn). An acid wash was performed to remove surface-bound transferrin that was not endocytosed. The Tf-A647 mean fluorescence intensity was measured by flow cytometry. The data correspond to the means ± SDs from duplicate experiments with at least 10,000 cells per condition. (D) Vero cells or SVG-A cells were prepared as described in the legends to Fig. 2C and D in the presence or absence of 50 μM ZCL278, and the amount of endocytosed particles (A647 positive and A568 negative) normalized by the amount of particles at the cell surface (A647 and A568 positive) was quantified by fluorescence microscopy. A total of 641 particles in the untreated control cells were counted and 698 particles in the ZCL278-treated cells were counted. DMSO, dimethyl sulfoxide. Error bars are the means ± SDs from two individual experiments.