The activity of the HIV-1 IRES is stimulated by oxidative stress and controlled by a negative regulatory element

Gendron, Karine; Ferbeyre, Gerardo; Heveker, Nikolaus; Brakier-Gingras, Léa

We investigated whether the activity of the 5 0 UTR IRES could be altered by a treatment with chemical agents causing a variety of stresses. We used hydrogen peroxide (H 2 O 2 ) and tert-butylhydroquinone (TBHQ) to induce oxidative stress, thapsigargin to induce an endoplasmic reticulum (ER) stress and deferoxamine (DFX), which is a hypoxia mimetic (Figure 3 ). We exposed to these agents either the wild-type IRES or the M4.stem143 mutant, where IRENE is altered and whose activity was increased compared to the wild-type. It is known that a treatment with these stress-inducing agents causes a decrease in cap-dependent initiation (53) . This was verified in our assays, using a [ 35 S]methionine labeling and it was observed that global translation, that reflects capdependent translation, was decreased by $2-fold under the different stress conditions investigated ( Figure 3A ). We measured the Fluc/RLuc ratio under these conditions. Rluc cannot be used to assess transient changes in cap-dependent initiation (54) because of its stability (half-life superior to 50 h) and its activity remained unchanged when the cells were exposed to the various stresses (see Figure 3B ). We observed that wild-type HIV-1 IRES activity, but not that of the M4.stem143 mutant, was increased $2-fold in presence of H 2 O 2 and TBHQ, which induce oxidative stress. No change in the IRES activity was seen with agents that induce either an ER stress or hypoxia. These results therefore suggest that the effect of mutating IRENE can be reproduced by an exposure of the wild-type IRES to oxidative stress.