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 perform a mutational analysis of the IRES located in the 5 0 UTR of HIV-1 full-length mRNA, a standard dual-luciferase reporter system was used (Figure 1B) , where the Rluc translation is cap-dependent whereas the Fluc translation depends on the HIV-1 5 0 UTR IRES. This reporter was similar to the reporter described by Brasey et al. (18) , who demonstrated the existence of the HIV-1 5 0 UTR IRES. The intercistronic region contains the complete 5 0 UTR, including the TAR and poly(A) stem-loops. It is well known that the presence of a thermodynamically stable stem-loop such as TAR interferes with readthrough, ribosomal scanning and reinitiation that could occur after Rluc termination of translation (36, 50) . Moreover, the Rluc coding sequence is terminated by three stop codons, which ensures efficient termination of translation. The initiation codon for Fluc is located within the HIV-1 5 0 UTR IRES. The 30 nt of HIV-1 RNA that follow this initiator codon and encode the beginning of the sequence of HIV-1 Gag were included in our construct, in order to maintain the integrity of the IRES. A sequence coding for a peptide linker was added between these 30 nt and the beginning of the Fluc coding sequence to avoid any interference of HIV-1 5 0 UTR with Fluc folding and activity. Jurkat T cells, a CD4+ T-cell line, were co-transfected with the dualluciferase reporter and a reporter coding for a shRNA targeting the Rluc coding sequence (51) . In the presence of a shRNA targeting Rluc, but not of a control shRNA, Rluc and Fluc expressions were decreased proportionally by $50%, confirming that Fluc is not expressed from a cryptic promoter or by spurious splicing ( Figure 1C ). The HIV-1 5 0 UTR IRES activity had been previously studied in HeLa cells (18, 38) . We worked with Jurkat T cells since they are closely related to the natural target cells of HIV-1. We observed here that the HIV-1 IRES is functional in Jurkat T cells, being $5-fold more active than a control construct where the 5 0 UTR was inserted in the opposite direction between the Rluc and Fluc coding sequences, and than a control construct where there is no IRES between the Rluc and Fluc coding sequences (data not shown). However, HIV-1 5 0 UTR IRES is weakly active, being $5-fold less efficient than the well-characterized HCV IRES (data not shown), an IRES known to be very active in a variety of cell lines (52) .