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

Wang, Qing S.; Jan, Eric

In contrast, FLuc enzymatic activity monitoring IAPV IGR IRES-mediated +1 frame translation was significantly affected in the presence of T2A [31] . Note that the T2A-containing +1 frame reporter constructs resulted in the synthesis of RLuc, the +1 frame cleaved proline-FLuc (P-FLuc) and ORFx-T2A proteins and the 0 frame *sORF2 ( Figure 2C, lane 1) . Calculation of the amount of P-Luc to the full-length ORFx-T2A-FLuc indicated .95% T2Amediated 'self-cleavage' activity ( Figure 2C ). By comparison of the [ 35 S]-methionine incorporation and the FLuc enzymatic activity in the Sf21 translation extracts, we have shown previously that the fusion of ORFx in-frame with the FLuc ORF inhibits FLuc enzymatic activity and that the inclusion of a functional T2A peptide into the bicistronic reporter system rescues the sensitivity of luciferase assay ( Figure 2C ) [31] . Inclusion of a mutant T2A peptide (D/E), which inactivates the 'self-cleaving' activity resulted in the full-length +1 frame ORFx-T2A-FLuc protein ( Figure 2C , lane 3) [31] .