Switch from Cap- to Factorless IRES-Dependent 0 and +1 Frame Translation during Cellular Stress and Dicistrovirus Infection

Wang, Qing S.; Jan, Eric

To test whether the +1 frame T2A containing construct can rescue luciferase enzyme sensitivity in vivo, in vitro transcribed capped bicistronic RNAs were transfected into Drosophila S2 cells. We previously reported that the luciferase activities of an IREScontaining bicistronic reporter RNA increased linearly up to 6 hours post transfection and the maximal luciferase activity occurred 6-10 hours post transfection [31] . These findings argue that the reporter RNA is intact and engaged in translation during the first 6 hours after transfection. Therefore, cells were collected and FLuc activities were detected at 6 hours post transfection ( Figure 2D ). As shown in vitro [31] , the IRES-dependent +1 frame FLuc luciferase signal was detected in S2 cells after transfection with a functional T2A-containing reporter RNA and not with a T2A-minus or mutant T2A (D/E) reporter RNA ( Figure 2D ). For the rest of these studies, we have used the T2Acontaining +1 frame reporter constructs to monitor +1 frame translation in vivo.