FIG. 5.

Analysis of the region of JEV NS5 that determines its IFN antagonist activity. (A) Schematic diagram of the full-length and truncated NS5-Flag-tagged fusion constructs and a summary of their IFN antagonist activities. (B) Protein expression patterns of NS5 deletion constructs. BHK-21 cells transfected with the indicated NS5 constructs for 24 h were harvested and analyzed by Western blotting with anti-Flag antibody. (C) Predicted molecular sizes of NS5 truncated mutants. (D) BHK-21 cells were cotransfected with pStat1-DsRed and various plasmids expressing the full-length or truncated NS5 proteins. At 24 h after transfection, cells were stimulated with IFN-αA/D (2,500 U/ml) for 16 h and then fixed and permeabilized for immunofluorescence assay. NS5 proteins were detected by using anti-Flag antibody and Alexa-Fluor 488 goat anti-mouse antibody (green). The locations of Stat1-DsRed (red) in the Flag-positive cells were observed under a fluorescence microscope. Representative cells from the same field for each experimental group are shown.