Fig. 1

Viral inclusions form in the absence of intersegment RNA–RNA interactions. 293 T cells were transfected for 16 h with the minimal protein components of an influenza vRNP: the three polymerase proteins (3P) (or, as a nonfunctional control, two polymerase proteins lacking PB2 - 2P) and NP, as well as with plasmids expressing GFP-NP and a a luciferase reporter plasmid cloned in negative sense and flanked by influenza promoter. mCherry-NP was also used instead of GFP-NP, when indicated. Luminescence was determined for luciferase expression and the values plotted as mean ± standard error of the mean (SEM). Results are a pool of 2 independent experiments; or b–e 293 T cells were further transfected with plasmids expressing vRNA from segments 7 and 8, or segment 7 alone, and a plasmid encoding NS2 when segment 7 was expressed alone. b Cells were lysed and the indicated proteins were detected by western blotting. c Cells were fixed and stained for Rab11 (red). White boxes show areas of co-localization between NP and Rab11. Nuclei are delineated by yellow dashed lines. Bar = 10 μm. d The frequency distribution of Rab11 inclusions within the three area categories (in μm²) was 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). Between 30 and 70 cells were analyzed per condition and 3 independent experiments were performed. e Duplicate samples were processed to detect segment 7 (red) and segment 8 (gray) RNA by FISH. White boxes show areas of co-localization between NP and viral segments. Nuclei are delineated by yellow dashed lines. Bar = 10 μm