Varicella Viruses Inhibit Interferon-Stimulated JAK-STAT Signaling through Multiple Mechanisms
Fig 5
ORF63 does not affect STAT2 activation but promotes IRF9-degradation.
(A) TRFs were infected with AdORF63 at MOI 20 and AdTA at MOI 10 in the presence of 1000 ng/ml Dox (control) or 1 ng/ml Dox for 48 hours. The cells were stimulated with 5000 U/ml uIFN for 20 min and lysates were analyzed via SDS PAGE and western blot for steady expression and phosphorylation status (p) of the indicated members of the JAK-STAT pathway. GAPDH was used as a loading control. ORF63 expression was confirmed using a specific antibody. One representative experiment out of three independent experiments is shown. The GAPDH signal was used a loading control. (B) TRFs were infected with AdORF63 at MOI 20 and AdTA at MOI 10 and simultaneously incubated with decreasing amounts of Dox. 48 hours p.i. the cells were stimulated with 5000 U/ml uIFN for 20 minutes, lysed and stained for IRF9, ORF63 and GAPDH. (C) Graph showing IRF9 and ORF63 expression normalized to GAPDH. Shown are the means ± standard error of two independent experiments. *: p-value < 0.005, **: p-value < 0.0005, ***: p-value < 0.00005; compared to expression in cells incubated with 1000 ng/ml Dox. (D) TRFs were infected with AdORF63 at MOI 20 and AdTA at MOI 10 and incubated with decreasing amounts of Dox. 48 hours p.i. the cells were lysed and stained for STAT1, STAT2, ORF63 and GAPDH. (E) Graphs showing STAT1 and STAT2 expression relative to GAPDH expression in the same samples. Shown are the means ± standard error of two independent experiments. NS: non-significant. (F) TRFs were infected like described in D and at 48 hours p.i. RNA was harvested to quantify IRF9 and ORF63 mRNA expression by qPCR. Data were normalized to the level of GAPDH mRNA expression in each sample. IRF9 expression is shown as delta cycle threshold (ΔCt). ORF63 expression is shown as relative expression compared to cells incubated with 1000 ng/ml Dox. Shown is a representative experiment of two independent experiments with three replicates/sample each.