Alternate strand DNA triple helix-mediated inhibition of HIV-1 U5 long terminal repeat integration in vitro.
Integration of the human immunodeficiency virus (HIV) DNA into the host genome is an obligatory process in the replicative life cycle of the virus. This event is mediated in vitro by integrase, a viral protein which binds to specific sequences located on both extremities of the DNA long terminal repeats (LTRs). These sites are highly conserved in all HIV genomes and thus provide potential targets for the selective inhibition of integration. The integrase-binding site located on the HIV-1 U5 LTR end contains two adjacent purine tracts on opposite strands, 5' . . . GGAAAATCTCT-3'/3'-CCTTTTAGAGA . . . 5', in parallel orientations. A single strand oligonucleotide 5'-GGTTTTTGTGT-3' was designed to associate with these tracts via its ability to form a continuous alternate strand DNA triplex. Under neutral pH and physiological temperature, the oligonucleotide, tagged with an intercalator chromophore oxazolopyridocarbazole, formed a stable triplex with the target DNA. The occurrence of this unusual triplex was demonstrated by both DNase I footprinting and electron microscopy. The triplex inhibits the two steps of the integrase-mediated reactions, namely, the endonucleolytic cleavage of the dinucleotide 5'-GT-3' from the 3' end of the integration substrate and the integration of the substrate into the heterologous target DNA. The midpoints for both inhibition reactions were observed at oligonucleotide concentrations of 50-100 nM. We believe that these results open new possibilities for the specific targeting of viral DNA LTR ends with the view of inhibiting integration under physiological conditions.
Bouziane
M
M
Laboratoire de Physicochimie et Pharmacologie des Macromolécules Biologiques CNRS URA 147, Institut Gustave Roussy, Rue Camille Desmoulins, 94805 Villejuif, France.
Cherny
D I
DI
Mouscadet
J F
JF
Auclair
C
C
D001483:Base Sequence; D018983:DNA Footprinting; D004279:DNA, Viral / Q000235:genetics; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D016662:Virus Integration
D004279:DNA, Viral; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides