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

Wang, Qing S.; Jan, Eric

We next investigated whether 0 and +1 frame translation directed by the IAPV IGR IRES are differentially regulated. It has been established that the IGR IRES can direct 0 frame translation in mammalian and yeast cells and can be stimulated under cellular stress conditions when cap-dependent translation is compromised [17, [38] [39] [40] [41] [42] [43] . To examine the extent to which the IGR IRES can direct 0 and +1 frame translation in S2 cells, we treated cells with different stressors, including DTT, pateamine A (PatA), or 4E1Rcat, each of which targets a specific step or activity in capdependent translation. DTT treatment induces ER stress and effectively leads to eIF2a phosphorylation and a shutdown of overall translation [27, 44] . PatA modulates eIF4A activity, thereby disrupting RNA helicase activity during cap-dependent translation [45] . 4E1Rcat binds to eIF4E and prevents the formation of the cap-binding complex [46] . The use of these compounds allows for systematic examinations into the cellular conditions that lead to IGR IRES translation. IRES translation was monitored by transfection of 59 capped bicistronic reporter RNAs in S2 cells treated with each stressor. Stressors were added to the cells at 1 hour after transfection and cells were harvested 5 hours later. As predicted, cap-dependent RLuc expression was inhibited by approximately 50% under DTT treatment ( Figure 5A ). In contrast, CrPV and IAPV IGR IRESmediated 0 frame translation were stimulated under DTT treatment ( Figure 5A ). Specifically, both IRESs stimulated 0 frame translation by approximately 2.5 fold ( Figure 5A ). Similar to that observed with the 0 frame translation, +1 frame translation increased to the same extent during DTT treatment ( Figure 5B ). The DPKI mutant IRES did not display significant FLuc expression under basal or DTT treatment, again confirming that FLuc expression is IGR IRES-dependent ( Figure 5B ). We found that the relative 0 and +1 frame translation did not appear to change under basal or DTT treatment ( Figure 5C ). These results suggest that +1 and 0 frame translation by the IGR IRES is regulated similarly during DTT treatment.