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 measure the IRES activity in the 5 0 UTR of HIV-1 group M subtype B full-length mRNA, we used a dual-luciferase reporter, pFRT-dual-IRES-HIV. This plasmid is derived from pcDNA5FRT (Invitrogen) where the KpnI and BamHI restriction sites of the original polycloning site were deleted to facilitate subsequent cloning of mutant IRESes. Our reporter pFRTdual-IRES-HIV contains the 5 0 UTR region of HIV-1 originating from pLAI, a vector expressing a molecular clone of HIV-1 group M subtype B proviral DNA (48) , inserted between the coding sequences of the Renilla luciferase (Rluc) and the firefly luciferase (Fluc). Expression of these genes is under control of a CMV promoter followed by a T7 promoter. The corresponding mRNA contains both the Rluc and the Fluc coding sequences. Rluc translation is cap-dependent and Fluc translation depends on HIV-1 5 0 UTR IRES. The initiator codon for Fluc expression is within the IRES and the context of the AUG (30 nt from the Gag precursor) was included in our constructs. Since the presence of these additional amino acids could affect Fluc activity, a sequence coding for a peptide linker (GGGGSGGGGS) was inserted by PCR before the Fluc coding sequence. The mutant IRESes were made by PCR amplification with four primers (49) . Mutants were named according to the position where the mutation/deletion starts in the 5 0 UTR of HIV-1 full-length mRNA according to pLAI. The details and the primers used for all these cloning experiments are found in the Supplementary Materials and Methods section.