Lipid mixing between single IAV particles and endosomes in control and IFITM3-expressing cells.

<p>(A–E) IAV particles co-labeled with AF488 (green) and vDiD (red) were pre-bound to A549-Vector or A549-IFITM3 cells in the cold and incubated at 37°C for 1 h. Particles exchanging vDiD with endosomes (arrows in A and D) exhibited marked increase in red signal. (A, B) Images of vDiD dequenching (extended projections) and particle fluorescence intensities obtained by tracking virions in A549 cells. A schematic illustration of IAV hemifusion with an endosome (gray), which leads to vDiD dequenching, is overlaid on the graph. I₁ and I₂ are fluorescence intensities immediately before dequenching and at the peak of dequenching, respectively. (C) IAV lipid mixing activity in A549-Vector cells is blocked in the presence of anti-HA antibody. AF488- and vDiD-labeled IAV were pre-incubated with 20 µg/ml of polyclonal anti-IAV antibody (Millipore, Billerica, MA) for 1 h at room temperature. Viruses were then bound to A549-Vector cells in the cold by spinoculation, and entry was initiated with warm imaging buffer supplemented with 20 µg/ml of the antibody. Images were collected from 12 fields and the average fraction of AF488 particles with the vDiD signal above the threshold level was determined and normalized to control conditions without the antibody. ***, P<0.001. (D, E) Representative images and analysis of lipid mixing in A549-IFITM3 cells. (F, G) Representative images and analysis of lipid mixing in MDCK-IFITM3 cells. The ratio of vDiD and AF488 signals (blue line) shows robust increase in the red signal in spite of variations in the green channel caused by axial displacement of the virus. Thick lines were obtained by smoothing raw fluorescence intensity data (thin lines). Cell contours are shown by dashed lines in A and D. See also corresponding <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004048#ppat.1004048.s013" target="_blank">movies S1</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004048#ppat.1004048.s014" target="_blank">S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004048#ppat.1004048.s015" target="_blank">S3</a>.