Inhibition of IFN-induced JAK-STAT signaling by VZV.

<p>(A) MRC5 cells were infected with VZV.eGFP (ratio 5:1) for 48 hours and stimulated for 20 minutes with 5000 U/ml uIFN. The lysates were analyzed for expression of STAT1, phosphorylated STAT1, STAT2, phosphorylated STAT2 (pSTAT2) and IRF9 using SDS-PAGE and western blot with specific antibodies. Lysates were stained for ORF31 and ORF63 to confirm viral infection and GAPDH was used as a loading control. (B/C) HEK 293T cells were co-transfected with plasmids encoding ISRE-luciferase and the indicated viral proteins. At 42 hours post transfection, the cells were stimulated with uIFN for 6 hours after which luciferase expression was measured. At this time part of the cells were lysed and expression of the transfected proteins was verified by SDS-PAGE and western blot using FLAG- and ORF63-specific antibodies. GAPDH was used as a loading control. (D) MRC5 cells and TRFs were co-infected with VZV AdORF63 at an MOI of 20, and AdTA at an MOI 10. The infected cells were incubated with the indicated concentrations of Dox. At 48 hpi the cells were stimulated with 5000 U/ml uIFN for 20 minutes. The lysates were analyzed for expression of STAT2, phosphorylated STAT2 (pSTAT2), IRF9, ORF63, and GAPDH by SDS-PAGE and western blot using specific antibodies.