Bouziane M, Cherny D I, Mouscadet J F, Auclair C
Laboratoire de Physicochimie et Pharmacologie des Macromolécules Biologiques CNRS URA 147, Institut Gustave Roussy, Rue Camille Desmoulins, 94805 Villejuif, France.
J Biol Chem. 1996 Apr 26;271(17):10359-64. doi: 10.1074/jbc.271.17.10359.
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.
人类免疫缺陷病毒（HIV）DNA整合到宿主基因组中是该病毒复制生命周期中的一个必需过程。这一事件在体外由整合酶介导，整合酶是一种病毒蛋白，它与位于DNA长末端重复序列（LTRs）两端的特定序列结合。这些位点在所有HIV基因组中高度保守，因此为选择性抑制整合提供了潜在靶点。位于HIV-1 U5 LTR末端的整合酶结合位点在相反链上包含两个两个相邻的嘌呤序列，5'...GGAAAATCTCT-3'/3'-CCTTTTAGAGA...5'，呈平行方向。设计了一条单链寡核苷酸5'-GGTTTTTGTGT-3'，通过其形成连续交替链DNA三链体的能力与这些序列结合。在中性pH和生理温度下，用嵌入剂发色团恶唑并吡啶咔唑标记的寡核苷酸与靶DNA形成了稳定的三链体。通过DNase I足迹法和电子显微镜都证明了这种不寻常三链体的存在。该三链体抑制整合酶介导反应的两个步骤，即从整合底物3'端进行二核苷酸5'-GT-3'的内切核酸酶切割以及底物整合到异源靶DNA中。在寡核苷酸浓度为50-100 nM时观察到两种抑制反应的中点。我们相信，这些结果为在生理条件下通过靶向病毒DNA LTR末端来抑制整合开辟了新的可能性。
