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

Wang, Qing S.; Jan, Eric

A 96-well plate was plated with equal amounts of protein (,100 mg total protein from S2 cells or 1 ml of in vitro translation reaction). 100 ml of Luciferase Assay Reagent II (Promega, Dual luciferase assay) was dispensed to determine FLuc activity using 10 second time intervals. Subsequently, 100 ml of Stop & Glo In vivo translation in S2 cells. In vitro transcribed 59 capped bicistronic reporter RNAs were transfected into Drosophila S2 cells. Cells were harvested at 6 hours, lysed and luciferase activity was measured. The white and black boxes represent RLuc and FLuc luciferase expression, respectively, indicative of cap-dependent and IRES-dependent translation. Relative luciferase activities (RLA), the quantitation of FLuc and RLuc enzymatic activity, and the relative ratios of FLuc/RLuc are normalized to that observed with the +1 frame T2A-containing reporter RNA. Shown are averages from at least three independent experiments (6 s.d.). doi:10.1371/journal.pone.0103601.g002 reagent (Promega, Dual luciferase assay kit) was dispensed to measure RLuc activity with the same time interval. A microplate luminometer (Berthold Technologies, Centro LB 960) supplied with software MikroWin 2000 was used to detect the luminescence signal, which is then normalized to obtain the relative luciferase activity (RLA). The quantitation of RLuc and FLuc activities were calculated separately and as a ratio of FLuc/RLuc.