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

To further investigate which portions of IRENE are involved in the control of HIV-1 5 0 UTR IRES activity, different mutations were made (described in Figure 2B and C). IRENE is formed by two stems separated by a 3A internal bulge on one side and a C bulge on the other side. The upper stem is itself interrupted by an AG bulge. We first disrupted either the lower stem (M3.stem134) or the upper stem (M4.stem143). We also made two mutants of the upper stem, M5.stem143up and M6.stem143bot, where either the top or the bottom part of the upper stem was disrupted, (see details in Figure 2 ). Finally, we deleted the 7-pyrimidine loop that caps this stem-loop (M7.Áloop151) or substituted the pyrimidines of this capping loop with purines (M8.loop151). Mutating the lower stem of IRENE did not influence the IRES activity (see M3.stem134) while disruption of the upper stem increased the IRES activity, as seen with M4.stem143 (200 ± 15%) ( Figure 2B ). An increase was also observed, although to a lesser extent, when only the top part of the upper stem was disrupted (M5.stem143up, 142 ± 7%), an effect similar to that obtained with M2.ÁSL134-178. Deleting or substituting the capping loop (M7.Áloop151 and M8.loop151) had no effect. Therefore, our results suggest that the negative determinant for the 5 0 UTR IRES activity seems to be located in the upper part of IRENE, but that the capping loop is not involved in this effect. We also investigated whether the 3A bulge that separates the upper from the lower stem in IRENE has an effect on the 5 0 UTR IRES activity. The 3A were substituted with either 3C, 3G or 3U. It was found that when the bulge is made of pyrimidines, the IRES Note that this figure describes the mutations that were introduced but not the structure adopted by the mutant stem-loops (see Figure 5 ). (C) Replacement of the 3A bulge of IRENE by 3C or 3U but not by 3G increases the IRES efficiency. The IRES activities for the mutants were assessed in lysates from Jurkat T cells transfected with the corresponding dual-luciferase plasmids. The Fluc/Rluc ratio obtained with the wild-type pFRT-dual-IRES-HIV (WT) was arbitrarily set at 100%. Results are the mean ± SEM of six independent experiments. The asterisk indicates mutants that were significantly different from WT (P < 0.05). efficiency is significantly higher (M9.CCC: 147 ± 5% and M11.UUU: 139 ± 5%) than when the bulge is formed by purines (WT with the 3A bulge: 100 ± 8% and M10.GGG: 114 ± 7%) ( Figure 2C ). Sequence alignment showed that IRENE is highly conserved in group M subtype B natural variants as mentioned in the above section. Most of the differences are located in the upper 7-pyrimidine loop, which does not influence the IRES activity. Twenty-one of the 97 variants analyzed have mutations in the upper stem, but only five of these mutations disrupt a base-pair. The 3A bulge is conserved in all these variants except three (see details in Supplementary Results section and Supplementary Figure S1A ).