Fig. 4

vRNP/Rab11 inclusions exchange material dynamically and form membraneless liquid organelles. A549 cells were transfected with a plasmid encoding GFP-NP and infected with PR8 virus, at an MOI of 5 for 10–16 h, and imaged under time-lapse conditions. a A representative cell is shown. The fluorescence signal of viral inclusions in this cell is depicted as: average intensity (in red), standard deviation (in green), the merge of both, and coefficient of variation. Two areas of viral NP inclusions, highlighted in purple and cyan boxes, were selected for fluorescence recovery after photobleaching (FRAP). Bar = 10 μm. b The photobleached regions are marked by a yellow circle. The black arrowhead indicates the time of photobleaching. Relative Fluorescence Intensity (R.F.I.) was plotted as a function of time for each particle. Images have been extracted from Supplementary Movie 16. c R.F.I. was plotted as a function of time for the means of 25 FRAP events (left graph). The means are shown (black) with error bars representing the standard deviation (gray). The percentage of recovery of each photobleached region is shown for specific times (right graph), with medians represented as red bars. A single experiment representative of two independent experiments is shown. d HeLa cells with the GFP-NP/PR8 system, infected for 16 h (MOI = 10), were imaged by confocal and electron microscopy, and the resultant images were superimposed. Areas of correlation, inclusions 1 to 3, are delineated by a dashed line in the upper panel and shown in greater detail in the lower panel. Progeny virions budding at the surface (black arrows) show that the cell is infected. Black delimited arrowheads show individual vesicles within the inclusion. e GFP-Rab11 WT cells were infected with PR8 virus (MOI = 5) for 16 h, and then stained for GFP (18 nm gold particles) and viral NP (6 nm gold particles). Inclusion areas are highlighted by black boxes. Black arrowheads indicate ER structures in the vicinity of viral inclusions. Black arrows show progeny virions budding at the cell surface. Black delimited arrowheads show vesicles. White arrowheads show electron-dense vRNPs. A single experiment representative of two independent experiments is shown