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

Translation studies in reticulocyte lysates showed that Gag (and implicitly Gag-Pol) can be expressed both by cap-and IRES-dependent modes but that the cap-dependent mode is predominant (35) . The full-length mRNA of HIV-1 group M subtype B, the group and type that predominates in infected patients of the Western World, contains an IRES in its 5 0 UTR (18). This IRES generates Gag (p55), initiating translation at the same initiation codon as the classical cap-dependent initiation. Another IRES was also found in the Gag coding sequence (17, 20) , which generates, in addition to Gag, a shorter isoform of Gag (p40). Brasey et al. (18) , using deletion mutants, delimited the boundaries of HIV-1 5 0 UTR IRES from nucleotides (nt) 104 to 336. They also showed that the portion encompassing nt 104-289 has an IRES activity very near that of the 104-336 segment. The 104-289 segment is present in all the RNA transcripts from HIV-1, suggesting that all these transcripts could be translated by an IRES-dependent mechanism in addition to the classical cap-dependent mechanism [see also ref. (29) ]. This is supported by recent studies from Charnay et al. (36) , showing that the mRNA coding for Tat can be translated by an IRES-dependent mechanism. Two proteins were found to modulate the activity of the 5 0 UTR IRES of HIV-1: the human embryonic-lethal abnormal vision (ELAV)-like protein HuR downregulates this IRES efficiency (37) , whereas the heterogenous nuclear ribonucleoprotein (hnRNP) A1 up-regulates its efficiency (38) . Interestingly, it was shown that infection with HIV-1 promotes relocalization of hnRNP A1 in the cytoplasm and enhances its expression, which then increases HIV-1 5 0 UTR IRES activity. It was also (31) . The regulatory motifs are indicated: the TAR structure (nt 1-57), the polyadenylation signal [poly(A)], the primer binding site (PBS), the dimerization initiation site (DIS), the major splice donor site (SD) and the packaging signal (c) (nt 312-325). The AUG initiator codon is boxed. (B) Representation of the dual-luciferase reporter, plasmid pFRT-dual-IRES-HIV. This plasmid contains the Rluc and the Fluc coding sequences, under control of a CMV and a T7 promoter, and separated by the 5 0 UTR region of HIV-1 full-length RNA (nt 1-369 based on pLAI). Translation of Rluc is cap-dependent while translation of Fluc depends on the 5 0 UTR IRES of HI1 RNA. The initiator codon for the Fluc coding sequence is within the 5 0 UTR IRES. An oligonucleotide coding for a peptide linker was inserted between the 30 nt from the Gag coding sequence that are present in the construct and the beginning of the Fluc coding sequence. (C) Effect of RNA interference against Rluc on the expression of Rluc and Fluc. Jurkat T cells were cotransfected with the dual-luciferase reporter and either pBS-U6-RLi that expresses a shRNA targeting Rluc or pBS-U6-ApaI that expresses a control shRNA. Jurkat T cells lysates were assayed for Fluc and Rluc activities 48 h post-transfection and these activities were normalized for the protein content. A value of 100% is ascribed to the Rluc and Fluc activity with the control plasmid. Results are the mean ± SEM of three independent experiments. observed that the La autoantigen binds to the 5 0 UTR of HIV-1 full-length RNA (39, 40) although a direct effect of La on the HIV-1 5 0 UTR IRES activity as an ITAF remains to be proven.