FIG 5.

Effect of NS1 mutations on segment 7 mRNA localization in infected cells. (A) 293T cells were infected with the indicated viruses at an MOI of 5, and at 6 h p.i. they were stained for segment 7 mRNA by FISH (green) and for DNA (4′,6′-diamidino-2-phenylindole; blue) before confocal imaging. Single optical slices are shown. Scale bar, 10 μm. (B) Individual cells were scored as to whether segment 7 mRNA staining was predominantly nuclear, cytoplasmic, or mixed. Values are the means ± standard errors of the means from three to six independent experiments. (C) Cell lysates were analyzed by Western blotting for the indicated antigens. (D) M1 and M2 accumulation from replicate experiments was quantified and expressed as a ratio relative to NP expression. Values plotted are normalized to the ratio seen with WT virus and are the means ± standard errors of the means of three independent experiments.