FIG. 3.

Analysis of C-terminal deletions of DEN NS4B. (A) Induction of ISRE-9-27-CAT reporter gene after treatment with IFN. Recombinant pCAGGS-HA plasmids containing each of the DEN-2 NS4B C-terminal deletions were transfected in Vero cells together with the ISRE-9-27-CAT plasmid, and CAT expression was stimulated with 1,000 U of IFN-β 24 h later. Results show the percentage of CAT activity for each individual construct. CAT activities were determined as mean values from two independent experiments (P values of 0.01 to 0.05). (B) Inhibition of NDV-GFP replication by IFN in the presence of DEN-2 NS4B derivatives. Vero cells were transfected with the plasmids expressing the indicated NS4B wild-type and mutant proteins and stimulated with 1,000 U of IFN-β prior to infection with NDV-GFP. Expression of GFP was visualized as green fluorescence by fluorescence microscopy. (C) STAT1 activation by IFN in the presence of NS4B derivatives. Vero cells simultaneously expressing NS4B (red) derivatives and GFP-STAT1 (green) were stimulated with 1,000 U of human IFN-β for 35 min, fixed, and permeabilized. HA-labeled protein was detected by fluorescence microscopy after incubation with polyclonal antibodies to HA and Texas red-labeled secondary antibody.