Fig. 4.

Enteroviral IRES reporter RNAs are inhibited by eIF2α phosphorylation. (A) HeLa TetON cells were transfected with luciferase (pTRE2-FL) reporters containing PV or coxsackievirus B3 (CVB3) IRES elements upstream of the firefly luciferase (FL) open reading frame (ORF). Expression of IRES-FL reporters and stress was coinduced for 3 h with constant exposure to tetracycline and Ars, respectively. Firefly luciferase (FL) activity is shown in relative light units (RLU). (B) In vitro-transcribed capped FL or uncapped IRES-FL reporter RNAs were electroporated into HeLa S3 cells prior to treatment with Ars for 90 min and analysis of FL expression. (C) Rabbit reticulocyte lysates supplemented with HeLa ribosomal salt wash initiation factors were pretreated with water or poly(I · C) for 30 min before being programmed with FL reporter RNAs. FL expression was measured after 45 min of translation at 34°C. (D) In vitro-transcribed capped FL or PV-FL reporter RNAs were electroporated into cells, which were then treated with 10 μm Sal003 for 90 min prior to analysis of FL expression. Results were compiled from three independent experiments, and error bars represent standard errors. (E) Immunoblots showing eIF2α or phospho-eIF2α (P-eIF2α) in the experiments above.