Fig. 6

Disruption of ER-Golgi trafficking disassembles vRNP hotspots. A549 cells were infected or mock-infected with PR8 virus at an MOI of 3. a At 90 min p.i., 2 μg mL⁻¹ of brefeldin A (BFA) or 10 μg mL⁻¹ nocodazole (NOC) were added and incubated for 10 h until the supernatant was collected and viral titres determined or cells were harvested in Laemmli’s and NP levels detected by western blotting. Statistical analysis of data was performed using a non-parametric one-way ANOVA, followed by Friedman’s multiple comparisons test (*p < 0.05). b–d At 8 hpi, cells were also treated or mock-treated with 2 μg mL⁻¹ of BFA for 1 h. b Cells were immunostained for the ER marker PDI (in green), the viral protein NP (in red) and the cis-Golgi marker GM130 (in gray) and imaged by confocal microscopy. Selected areas of the cytoplasm are marked by white boxes and displayed on the top left corner of the images. Bar = 10 μm. c The frequency distribution of NP inclusions within the three size categories (in μm²), the number of inclusions per μm², and the percentage of NP staining that is inside inclusions were plotted for each condition. Statistical analysis of data was performed using a non-parametric Kruskal–Wallis test, followed by Dunn’s multiple comparisons test (***p < 0.001). An average of 60 cells from 2 independent experiments was analyzed per condition. d Infected cells were stained for the host protein Rab11 (in green) and the viral proteins NP (in red) and M2 (in gray). Cells were imaged by confocal microscopy. Areas highlighted by the white box are shown on the right top corner of each image. e A549 cells were transfected with a plasmid encoding GFP-NP and co-infected with PR8 virus, at an MOI of 5, for 10 h. Cells were imaged under time-lapse conditions in the absence or immediately after adding 2 μg mL⁻¹ of BFA. Bar = 10 μm. Images were extracted from Supplementary Movies 20 and 21 , and are representative of 9 videos from 2 independent experiments