FIG. 4.

NS1 protein expression inhibits dsRNA- but not arsenite-induced activation of c- Jun. (A) MDCK cells were transfected with a plasmid expressing GST-tagged JNK/SAPKβ together with either empty vector (lanes vector) or derivatives thereof expressing NS1 WT, NS-IAmut1, or the NS1-R38A/K41A RNA-binding mutant protein (lanes WT, IAmut1, and R38A K41A, respectively). At 24 h later, the cells were left untreated or stimulated with synthetic dsRNA (50 μg/ml) for 6 h and extracts were prepared as described previously (30). JNK activity was assessed in the lysates by immune complex kinase assays using GST-c-Jun(1-135) as a substrate (30). The amount of GST-JNK in each sample was determined by immunoblotting with GST-specific antiserum. (B) In parallel reactions, we analyzed c-Jun phosphorylation in cells that were transfected with empty or NS1 expression vector and were mock treated (lane 0) or stimulated with 0.5 mM sodium arsenite, a common JNK activator, for 30 min (lanes 0.5).