Subcellular distribution of NS5 and dsRNA in WT and mutant DENV transfected cells. BHK-21 cells were transfected with DENV2 WT (A), R888K (B), and Y838F (C) mutants as in Figure 1B and analyzed at 72 h post-transfection for NS5 (green) and dsRNA (red) by immunofluorescence assay. Digitized images were captured by Zeiss LSM710 upright confocal microscope using a 63× oil immersion lens. The % infectivity is indicated, and the insets in the bottom panes show the zoom-in views of the dotted box regions. Two independent experiments were performed, and the representative images were observed for more than 99% of the cells. Intensity quantification of (D) NS5 and (E) dsRNA fluorescence of WT, R888K, and Y838F infected cells. Data are presented as bar graphs with average ± SD obtained from n = 30 infected cells (nine field images for WT, 24 field images for R888K, and 15 field images for Y838F). Values indicated on top of the bars of graph in D are the % infectivity of the respective viruses. Difference in fluorescence intensity between WT and R888K or Y838F was compared by Student's t-test and a P-value <0.05 was considered significant (*) P < 0.05.
