Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

Spence, Jennifer S.; Krause, Tyler B.; Mittler, Eva; Jangra, Rohit K.; Chandran, Kartik

Cells were seeded on fibronectin-coated 35-mm-diameter coverslip dishes (MatTek) 24 h in advance of experiments and were grown to approximately 70% confluence. Dishes were placed on ice for several minutes before addition of virus. Virus in imaging buffer (140 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 20 mM HEPES, 5 mM glucose, 2 g/ml Hoechst stain, and 2% FBS, pH 7.4) was spinoculated onto cells by 20 min of centrifugation at 1,500 ϫ g at 4°C. Inoculum volumes were adjusted so that 200 to 400 cell-associated particles were visible per field. Unbound particles were removed by four washes with cold PBS, and 500 l cold imaging buffer was added to the dish. Samples were mounted on the microscope and quickly focused, and then 1.5 ml warm imaging buffer was added to start the experiment (t ϭ 0). Images were acquired every 4 s over the duration of the experiments using a single Z-section, as the cells were very flat. Excluding the initial kinetics studies, all experiments were limited to 2 h. Data analysis. Image analysis and single-particle tracking were performed using Volocity software (PerkinElmer). Excluding minor adjustments in brightness and contrast, image files were not manipulated. For single-particle dequenching measurements, puncta were thresholded by initial intensity and size. Puncta falling outside the range of 0.125 to 1.0 m 2 expected of discrete VSV particles were excluded from analysis.