Cherepanov P, Surratt D, Toelen J, Pluymers W, Griffith J, De Clercq E, Debyser Z
Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium and The Lineberger Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295, USA.
Nucleic Acids Res. 1999 May 15;27(10):2202-10. doi: 10.1093/nar/27.10.2202.
Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA into the genome of a human cell is an essential step in the viral replication cycle. Understanding of the integration process has been facilitated by the development of in vitro assays using specific oligonucleotides and recombinant integrase. However, understanding of the biology of retroviral integration will require in vitro and in vivo model systems using long DNA substrates that mimic the HIV cDNA. We have now studied the activity of recombinant HIV-1 integrase on a linear 4.7 kb double-stranded DNA, containing flanking regions of approximately 200 bp that represent the intact ends of the HIV-1 long terminal repeat (LTR) sequences (mini-HIV). The strand transfer products of the integration reaction can be directly visualized after separation in agarose gels by ethidium bromide staining. The most prominent reaction product resulted from integration of one LTR end into another LTR end (U5 into U5 and U5 into U3). Sequence analysis of the reaction products showed them to be products of legitimate integration preceded by correct processing of the viral LTR ends. Hotspots for integration were detected. Electron microscopy revealed the presence of a range of reaction products resulting from single or multiple integration events. The binding of HIV-1 integrase to mini-HIV DNA was visualized. Oligomers of integrase seem to induce DNA looping whereby the enzyme often appears to be bound to the DNA substrate that adopts the structure of a three-site synapsis that is reminiscent of the Mu phage transposase complex.
人类免疫缺陷病毒1型（HIV-1）的cDNA整合到人类细胞基因组中是病毒复制周期中的关键步骤。利用特定寡核苷酸和重组整合酶开发的体外检测方法有助于人们对整合过程的理解。然而，要深入了解逆转录病毒整合的生物学机制，还需要使用模拟HIV cDNA的长DNA底物构建体外和体内模型系统。我们现在研究了重组HIV-1整合酶对线性4.7 kb双链DNA的活性，该双链DNA含有约200 bp的侧翼区域，代表HIV-1长末端重复序列（LTR）的完整末端（微型HIV）。整合反应的链转移产物在琼脂糖凝胶中分离后，通过溴化乙锭染色可直接观察到。最显著的反应产物是一个LTR末端整合到另一个LTR末端（U5到U5以及U5到U3）。对反应产物的序列分析表明，它们是在病毒LTR末端经过正确加工后进行合法整合的产物。检测到了整合热点。电子显微镜显示存在一系列由单次或多次整合事件产生的反应产物。观察到了HIV-1整合酶与微型HIV DNA的结合。整合酶寡聚体似乎会诱导DNA成环，在此过程中，该酶常常与呈现三点联会结构的DNA底物结合，这种结构类似于Mu噬菌体转座酶复合物。
