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 characterize the regulation of the HIV-1 5 0 UTR IRES activity, we also investigated substitution mutations that were found by Abbink et al. (55) to influence the BMH-LDI switch (see Introduction section) (Figure 4) . Two of the six mutations investigated, M12.DIS240 and M16.loopI302, influenced the 5 0 UTR IRES activity, decreasing its value to $60% of wild-type efficiency. These mutations consisted, respectively, of a substitution of three purines with pyrimidines in a 4-nt bulge in the bottom part of the DIS hairpin and in a substitution of four A with pyrimidines in the beginning of loop I, downstream of the SD hairpin. When the purines directly opposite to the 4-nt bulge in the DIS hairpin were substituted with pyrimidines (M13.DIS278), when the two purines located at the end of loop I were Figure 3 . HIV-1 5 0 UTR IRES activity is increased in presence of agents that induce oxidative stress. Jurkat T cells were exposed to different agents [DMSO: control (6 h), H 2 O 2 : hydrogen peroxide (4 h), thaps: thapsigargin (6 h), TBHQ: tert-butylhydroquinone (6 h) and DFX: deferoxamine (8 h) ]. (A) The cap-dependent translation is decreased following exposure to the different agents, as shown by metabolic labeling with [ 35 S]methionine and measurement of the radioactivity incorporated into trichloroacetic acid-precipitable material. Radioactive counts were normalized for total protein content. A value of 100% was arbitrarily ascribed to the control sample (DMSO). (B) The activity of WT and M4.stem143 mutant IRES was assessed in cell lysates as described in the legend to Figure 2 . Results show the Fluc/Rluc ratio and the Fluc and Rluc activities. A value of 100% was arbitrarily ascribed to WT. Results are the mean ± SEM of three independent experiments. The asterisk indicates the treatments that significantly increase the activity of the WT IRES (P < 0.05). The activity of the mutant IRES was not significantly altered by exposure to the different agents. substituted with pyrimidines (M17.loopI310) or when the SD hairpin was mutated (M14.SD296 and M15.SD293), there was no effect on the IRES activity. It was also observed that, in M15.SD293, the sequence of the splicing donor site in the 5 0 UTR of HIV-1 RNA is mutated, but the expression of Fluc in this construct is not affected (data not shown), in line with the fact that Fluc expression results from a genuine IRES activity and not from a splicing event (see above). The 4-nt bulge in the DIS hairpin is highly conserved, but the A stretch in loop I shows some variability among natural variants of HIV-1 group M subtype B (see Supplementary Results section and Supplementary Figure S1C and D) .