FIG. 4.

Reduction of IFN-α-stimulated Stat1 and Tyk2 tyrosine phosphorylation in Vero cells infected with a recombinant Sindbis virus expressing JEV NS5. (A) Schematic representation of a recombinant SIN expressing JEV NS5 protein. The construct, including a respiratory syncytial virus (RSV) promoter-driving expression cassette, was able to transcribe and translate the SIN replicase complex from nonstructural genes (nsP1, nsP2, nsP3, and nsP4). The replicase produced then activates the subgenomic promoter (SP) to drive the expression of SIN structural genes and JEV NS5. (B) Vero cells were mock infected or infected with wt-SIN, recombinant NS5-SIN, or JEV (MOI = 10) for 6 h. The cells were then stimulated with IFN-αA/D (1,000 U/ml) for 30 min or left unstimulated. The cell lysates were harvested for Western blotting with anti-phospho-Stat1 (Tyr701), anti-Stat1, anti-Flag, and anti-JEV NS3 antibodies, as indicated on the right sides of the gels. (C) Vero cells were prepared as described for panel B except that cells were stimulated with IFN-αA/D for 15 min before harvesting for Western blotting with anti-phospho-Tyk2 (Tyr1054/1055), anti-Tyk2, anti-Flag, and anti-JEV NS3 antibodies as indicated.