ANALYSIS OF THE HIV INTEGRATION REACTION USING RECOMBINANT INTEGRASE

HIV整合酶(IN)是病毒整合所必需的一种病毒酶 DNA进入宿主染色体。这种类型的集成是高度 针对逆转录病毒，因此HIV-1IN是 抗病毒治疗。破坏整合酶功能也是可取的 在艾滋病毒减毒活疫苗的设计中。我们已经表达了IN基因 在大肠杆菌中以全长和截短形式存在。杂交瘤已经被 用全长克隆制备的单抗与 Western印迹结果显示HIV-1不表达HIV-2。In具有特定的DNA裂解 活性和非特异性DNA结合活性。我们之前已经展示过 只有IN的C末端能够与DNA结合 西南部的污点。一系列C端缺失克隆的构建 对蛋白质的分析表明，DNA结合域 可以定位在180-248个氨基酸范围内。此区域包含 一系列碱性氨基酸(赖氨酸和精氨酸)。站点定向 其中一些残留物的突变现在已经完成，结果是 突变蛋白正在进行提纯，并将对其DNA进行分析 结合能力以及在特定的寡核苷酸裂解试验中。 这组最新的克隆是在谷胱甘肽转移酶中构建的 融合载体，提供了一种通过亲和纯化的简化方法 层析，以及另外，生产标签的可能性 用于蛋白质-蛋白质关联研究的蛋白质。In一直在 假设在体内起二聚体的作用，但蛋白质的区域 此交互所需的信息未知。我们还提纯了大型 David Davies用于生成FAB的IN MAb的数量 用于IN的X射线结晶学的碎片；尝试完全结晶- Long In本身到目前为止还没有成功，因为它倾向于 聚集在一起。这可能最终促进酶抑制剂的设计，通过 提供有关IN~S三维结构的信息。

HIV integrase (IN) is a viral enzyme required for integration of virus DNA into the host chromosome. This type of integration is highly specific for retroviruses, and HIV-1 IN is therefore a target for antiviral therapy. Disruption of integrase function is also desirable in design of live attenuated HIV vaccines. We have expressed the IN gene in E. coli in both full-length and truncated forms. Hybridomas have been prepared using the full-length IN clone and the MAbs are reactive with HIV-1 but not HIV-2 by Western blot. IN possesses a specific DNA cleavage activity and a non-specific DNA-binding activity. We have previosly shown that only the C-terminus of IN was capable of binding DNA in a Southwestern blot. Construction of a series of C-terminal deletion clones and analysis of the proteins demonstrated that the DNA binding domain could be localized to within amino acids 180-248. This region contains an array of basic amino acids (lysines and arginines). Site-directed mutagenesis of some of these residues has now been done and the resultant mutant proteins are being purified and will be analysed for their DNA binding ability as well as in a specific oligonucleotide cleavage assay. This newest set of clones was constructed in a gluathione transferase fusion vector, which provides for a simplified purification by affinity chromatography, and additionally, the possibility of producing labelled proteins for use in protein-protein association studies. IN has been postulated to act as a dimer in vivo, but the region of the protein required for this interaction is unknown. We have also purified large quantities of the IN MAb for David Davies to use to generate Fab fragments for X-ray crystallography of IN; attempts to crystallize full- length IN alone have thus far not been successful due to its tendency to aggregate. This may eventually facilitate design of enzyme inhibitors by providing information about IN~s 3-dimensional structure.