Figure 4

Sindbis vector infection activates JNK. (A) JNK phosphorylation is induced by infection with Sindbis vector. Lysates of SV (+) infected or mock (-) infected MOSEC cells were collected at 2, 4 and 6 h.p.i. Immunoblotting with phospho-JNK first detects phosphorylation at 4 h.p.i. (B) Cell permeable JNK inhibitory peptide inhibits c-jun phosphorylation. MOSEC cells were treated for 1 hour with JNK specific inhibitory peptide and then infected with SV or mock infected and subjected to a JNK specific kinase assay 24 h.p.i. (C) Inhibition of JNK activation has no effect on Sindbis-induced translational arrest. MOSEC cells treated with JNK inhibitor and infected were labeled with ³⁵S methionine 24 h.p.i. (D) JNK inhibition results in an increase in cell viability following Sindbis infection. MOSEC or Pan02 cells pretreated for 1 hour with a JNK-specific peptide inhibitor and then infected with SV or mock infected were assessed for cell viability 24 h.p.i. Data in D represents the SEM (error bars) of three experiments. Each sample was compared to the non-treated infected control at the same time point. Statistical significance was calculated by a two-tailed student t-test (** P < 0.005). (E) JNK phosphorylation is downstream of PKR activation. Lysates from MOSEC or Pan02 cells, transfected with siPKR or siGLO (control) and infected with SV or mock infected, were collected at 8 h.p.i. Immunoblotting for phosphorylated JNK indicates that it remains dephosphorylated in cells treated with siPKR. The immunoblots in A&E were stripped and reprobed for β actin as a loading control.