FIG 2.

Importance of the actin network during JUNV entry. (A, B) Vero, SUM159, SVG-A, or A549 cells were untreated or pretreated for 15 min with 1 μM latrunculin A (Lat A), 1 μM latrunculin B (Lat B), 1 μM cytochalasin D (Cyto D), or 1 μM jasplakinolide (Jasp) and subsequently infected with JUNV in the presence or absence of the drugs for an additional 30 min. Then, the cells were washed and incubated for 16 h in virus medium containing an anti-GP neutralizing antibody. (A) The cells were fixed and stained with an anti-NP antibody coupled to A647, and the percentage of infected cells was measured by flow cytometry. The data are normalized to the percentage of infected untreated cells for each cell type. Error bars are the means ± SDs from duplicate experiments with at least 10,000 cells per condition. (B) Cell viability was assessed using the CellTiter-Glo viability assay with cells prepared in parallel with the cells in the assay for which the results are presented in panel A. Error bars are the means ± SDs from triplicate experiments. Non-inf, noninfected. (C, D) Vero cells were pretreated in the presence or absence of Lat B for 15 min and subsequently infected with JUNV-A647 for 30 min in the presence or absence of Lat B. The cells were then fixed and stained with an anti-GP antibody (GB03) coupled to A568 without permeabilization. (Left and middle) Fluorescence from individual channels; (right) overlay of the fluorescence from the GP-A568 (green) and JUNV-A647 (red) channels together with the DAPI (4′,6-diamidino-2-phenylindole) signal (blue). Red arrowheads, a JUNV particle that has been endocytosed (A647 positive and A568 negative); yellow arrowheads, two particles that are still at the surface of the cell (A647 and A568 positive). (D) Quantification of the amount of A647-positive and A568-negative particles (endocytosed) normalized by the amount A647- and A568-positive particles (at the cell surface). A total of 124 particles over 26 fields of view were counted for the untreated control, and 42 particles over 16 fields of view were counted for cells treated with 1 μg ml⁻¹ Lat B. Error bars are the means ± SDs from two individual experiments.