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

Wang, Qing S.; Jan, Eric

DPKI IRES did not result in expression of either 0 or +1 frame translation products, again confirming that IRES translation was being assayed ( Figure S1 , C lane 4). The ratio of FLuc/RLuc was measured either by quantitiating [ 35 S]-methionine incorporation or by measuring the enzymatic luciferase activity (Figure S1, D and E). IGR IRESmediated 0 frame FLuc enzymatic activity was not significantly affected in the presence or absence of the T2A peptide ( Figure S1 , E). Thus, for most of the studies, 0 frame translation directed by the IGR IRES was monitored using the T2A-less reporter constructs. To confirm that IRES translation was monitored using the coupled transcription-translation Sf21 system, we incubated in vitro transcribed 59capped reporter RNA ( Figure S1 , F) in the Sf21 translation extract system. Capped reporter RNAs were obtained by using in vitro transcription reactions in the presence of cap analog m 7 G(59)ppp(59)G [31] . As expected, reporter RNAs containing the mutant DPKI IRES did not result in expression of the FLuc activity.