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 hos t 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 D NA long terminal repeats (LTRs). These sites are highly conserved in a ll HIV genomes and thus provide potential targets for the selective in hibition 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'-CCTTTTA- GAGA.,,5', in parallel orientations. A single strand oligonucleotide 5'-GGTTTTTGTGT-3' was designed to ass ociate with these tracts via its ability to form a continuous alternat e strand DNA tripler. Under neutral pH and physiological temperature, the oligonucleotide, tagged with an intercalator chromophore oxazolopy ridocarbazole, formed a stable tripler with the target DNA. The occurr ence of this unusual tripler was demonstrated by both DNase I footprin ting and electron microscopy. The tripler inhibits the two steps of th e integrase-mediated reactions, namely, the endonucleolytic cleavage o f the dinucleotide 5'-GT-3' from the 3' end of the integration substra te and the integration of the substrate into the heterologous target D NA. The midpoints for both inhibition reactions were observed at oligo nucleotide concentrations of 50-100 nM. We believe that these results open new possibilities for the specific targeting of viral DNA LTR end s with the view of inhibiting integration under physiological conditio ns.