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

The effect of IRENE on the IRES activity is more complex. Our results show that the IRES activity is weaker in the presence of IRENE than in its absence, but that mutations in the upper stem of IRENE increase the IRES activity. Moreover, inducing oxidative stress increases the activity of the wild-type IRES but not that of the M4.stem143 mutant, which is more active than the wild-type. Figure 5 shows the secondary structure of wildtype and mutated IRENE, determined by the standard mfold algorithm. The secondary structure of IRENE is quasi-identical in the different structures that were proposed for the 5 0 UTR of HIV-1 full-length RNA (31) and the structure of IRENE determined by mfold is identical to that found in the Weeks conformation that we used throughout this study. The structure of IRENE for the M3.stem134, M6.stem143bot, M7.Áloop151 and M8.loop151 mutants is very similar to that of the wild-type ( Figure 5A and C), conserving the same orientation of the bend induced by the 3A bulge separating IRENE in two parts. These four mutants have an IRES activity comparable to that of the wild-type IRES and it could be suggested that the wild-type IRES and these mutants adopt a weakly active conformation, which could be stabilized by the binding of a negative factor. As to the mutants with an increased IRES activity due to either the deletion or a mutation in the upper stem of IRENE (M2.ÁSL134-178, M4.stem143, M5.stem143up), they have a different conformation for IRENE, but this is consistent with the fact that a variety of IRES structures can recognize the ribosome (3). Finally, substituting the 3A bulge with 3G in IRENE did not change the IRES activity while replacing the 3A with pyrimidines increased the IRES activity $1.5-fold (see Figure 2C ). These 3A are poorly reactive according to Wilkinson et al. (31) and could thus be involved in RNA-RNA contacts with other parts of the IRES, which could contribute to a weakly active conformation of the IRES. Such contacts would be maintained when the 3A are replaced by 3G but not by pyrimidines.