FIG 8.

Differential requirements for RACK1 in JNK activation during poxvirus infection versus ribotoxic stress responses. (A) HAP1 parental and RACK1 KO cells were treated with anisomycin (ANS) (10 μM) for 1 h or infected with VacV at MOI 25 for 24 h. The cells were lysed and subjected to Western blotting with the indicated antibodies. ANS-induced JNK activation is completely dependent on RACK1, while VacV-induced JNK activation is not. (B) Validation of JNK inhibitor VIII efficacy in cells treated with ANS or infected with VacV. JNKVIII blocks JNK activity but not its phosphorylation. To verify inhibitory efficacy, HAP1 cells were treated with dimethyl sulfoxide (DMSO) solvent control or ANS (10 μM) for 1 h or infected with VacV at MOI 25 for 24 h in the presence of JNK inhibitor VIII at the indicated concentrations. Uninfected or infected HAP1 cells were treated with DMSO as a solvent control. Phosphorylated c-Jun was used as a readout for inhibition of JNK activity. (C) JNK activity is not required for VacV protein synthesis. HAP1 cells were infected with VacV as described for panel B. Representative early-intermediate (I3) and late (F17, A14) VacV proteins were analyzed by Western blotting. Early and late viral protein levels were unaffected by JNK inhibition. The results are representative of three independent experiments.