Fig. 1

Effect of mutations in the NS1 effector domain on segment 7 mRNA localisation. a–c 293T cells were transfected with plasmids to recreate segment 7 RNPs or, as a negative control with a combination that excluded a plasmid expressing PB2 (2PNP), together with the indicated NS1-GFP polypeptides. a Cells were processed for FISH analysis at 24 h post transfection (p.t.) using Cy3-labelled RNA probes for detection of positive sense segment 7 viral RNAs (red). GFP fluorescence was also detected (green). Images were captured using a Leica-TCS confocal microscope and Leica TCS analysis software. b Duplicate cell samples were lysed and examined by SDS-PAGE and western blotting for GFP and tubulin. c Individual cells were scored according to the predominant cellular localisation of segment 7 mRNA considering three phenotypes: nuclear, cytoplasmic or mixed. Values are the mean ± range from two independent experiments. A 2-way ANOVA was used to test for statistically significant differences between the values for each category from those obtained for WT-NS1 (NS, not significant; ****p < 0.0001). d 293T cells were mock infected or infected with the indicated viruses at an M.O.I. of 5. At 6 h post infection (p.i.), cells were processed for FISH as above (red) and stained with anti-NS1 serum (green) and DAPI (for DNA; blue). Scale-bars indicate 10 µm. Lanes 1 and 2 in b and a portion of the corresponding numerical data in c are taken with permission from Figure 4b, c respectively of [4]