Written informed consent was obtained from the parents of the patient for publication of this report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. The activity of the HIV-1 IRES is stimulated by oxidative stress and controlled by a negative regulatory element Initiation of translation of the full-length messenger RNA of HIV-1, which generates the viral structural proteins and enzymes, is cap-dependent but can also use an internal ribosome entry site (IRES) located in the 5′ untranslated region. Our aim was to define, through a mutational analysis, regions of HIV-1 IRES that are important for its activity. A dual-luciferase reporter construct where the Renilla luciferase (Rluc) translation is cap-dependent while the firefly luciferase (Fluc) translation depends on HIV-1 IRES was used. The Fluc/Rluc ratio was measured in lysates of Jurkat T cells transfected with the dual-luciferase plasmid bearing either the wild-type or a mutated IRES. Deletions or mutations in three regions decreased the IRES activity but deletion or mutations of a stem-loop preceding the primer binding site increased the IRES activity. The wild-type IRES activity, but not that of an IRES with a mutated stem-loop, was increased when cells were treated with agents that induce oxidative stress. Such stress is known to be caused by HIV-1 infection and we propose that this stem-loop is involved in a switch that stimulates the IRES activity in cells infected with HIV-1, supporting the suggestion that the IRES activity is up-regulated in the course of HIV-1 replication cycle. coding sequences. This PCR introduced three stop codons to terminate the Rluc coding sequence. The rest of the 5'UTR was amplified directly from a proviral molecular clone of HIV-1 group M subtype B (pLAI) (4) . The initiation codon for Fluc expression is located within the 5'UTR IRES and the context of the AUG encompassing 30 nt from the Gag sequence was included. A peptide linker (GGGGSGGGGS) was inserted by PCR immediately upstream of the Fluc coding sequence. The first half of the linker was inserted using PCR amplification on pLAI with the 5'UTR-AflII(+) and 5'UTR-BamHI(-) primers and the second half of the linker with the 5'UTR(+) and Linker-BamHI(-) primers. The linker was cloned in the AflII and BamHI restriction sites of pDual-HIV(-1)ΔAflII-TAR to generate pDual-IRES-HIV. This last step removed the frameshift region originally present in pDual-HIV(-1). Finally, we cut the fragment containing the Rluc coding sequence, the 5'UTR region of HIV-1 RNA and the Fluc coding sequence, using PmeI and ApaI restriction sites. This fragment was inserted into pcDNA5FRT (Invitrogen) previously linearized with SciI and ApaI to produce pFRT-IRES-HIV. Prior to this last cloning step, the KpnI and BamHI restriction sites from pcDNA5FRT were eliminated to facilitate subsequent cloning of mutant IRESes. To this end, an oligonucleotide cassette formed by the K7ΔKpnIΔBamHI(+) and