Fig. 2.

Apilimod and Vacuolin-1 inhibit VSV-MeGFP-ZEBOV. (A) Schematic of entry assay where SVG-A cells were infected with VSV-MeGFP (MOI = 4), VSV-MeGFP-V269H (MOI = 4), or VSV-MeGFP-ZEBOV (MOI = 4). Experiments were performed in the presence of 5 µg/mL cycloheximide (CHX) to prevent protein synthesis. Entry assay was based on the appearance of MeGFP fluorescence on the nuclear margin, on a per cell basis, of fixed infected cells visualized by fluorescence microscopy. Staining the fixed cells with Alexa647-labeled wheat germ agglutinin identified the plasma membrane of each cell (dashed outlines in C). (B) Virus infection in the absence of CHX (Left) resulted in the appearance of MeGFP fluorescence throughout the cell volume. The presence of CHX resulted in virus entry being observed by MeGFP fluorescence at the nuclear margin, which was released from incoming viral particles (Right, white arrows). (Scale bar: 10 µm.) (C) Representative examples of maximum-Z projections images from the whole-cell volume obtained with optical sections separated by 0.3 µm using spinning disk confocal microscopy. MeGFP fluorescence at the nuclear margin released from incoming viral particles is highlighted (white arrows). (Scale bar: 10 µm.) (D) Quantification of the number of cells with nuclear margin labeling from three independent experiments, each determined from fields containing 59 to 90 cells (error bars show SEM). The statistical significance of the entry data was analyzed for statistical significance by one-way ANOVA and Tukey post hoc test (***P ≤ 0.001).