ANALYSIS OF THE HIV INTEGRATION REACTION USING RECOMBINANT INTEGRASE

HIV整合酶(IN)是整合病毒DNA所必需的一种病毒酶 进入宿主染色体。这种类型的集成高度特定于 因此，HIV-1IN是抗病毒治疗的目标。 在LIVE设计中也希望破坏整合酶功能 减毒艾滋病毒疫苗。我们已经在大肠杆菌中表达了IN基因 融合蛋白。克隆的IN对HIV-1和HIV-2阳性均有反应 与患者血清进行酶联免疫吸附试验，兔抗重组蛋白抗血清 免疫印迹显示只与HIV-1 IN反应，而不与HIV-2 IN反应。杂交瘤 用IN表达克隆制备了具有反应性的单抗 经Western印迹分析，该蛋白与HIV-1融合，但不与HIV-2融合。一份发明报告已归档 一份手稿正在准备中。表达N-和的其他克隆 已经构建了IN的C-末端半部分，并表达了它们的蛋白质 使用西南印迹程序检查了结合DNA的能力。 完整的IN分子和C-末端蛋白与DNA结合；N- 末端部分没有结合活性，表明C- 末端区域包含DNA结合位点。提纯全长 重组IN在体外特异的酶活性测定中显示出活性 涉及从相应的寡核苷酸中切割2个碱基对的活性 HIV-1 LTR.为了更准确地定位C基因中的DNA结合位点 分子末端的一半，我们已经构建了一系列额外的 亚克隆并对缺失的蛋白质进行DNA结合分析。这有 使我们能够将DNA结合位点映射到180-248个氨基酸。一份手稿 正在Nucl出版。酸的研究和制备的额外删除是 正在进行中。我们还与美国国立卫生研究院的大卫·戴维斯合作 为他提供了大量的In Mab，用于Fab的生成 IN的X射线结晶学碎片，这最终可能有助于 酶抑制剂的设计。

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 as a fusion protein. The cloned IN is reactive with both HIV-1 and HIV-2 positiv patient sera in ELISA, while rabbit antisera to the recombinant protein are reactive only with HIV-1 IN but not HIV-2 IN by Western blot. Hybridomas have been prepared using the IN expressing clone and the MAbs are reactive with HIV-1 but not HIV-2 by Western blot. An invention report has been file and a manuscript is in preparation. Additional clones expressing the N- and C-terminal halves of IN have been constructed and the proteins they express examined for ability to bind DNA using a Southwestern blotting procedure. The complete IN molecule as well as the C-terminal protein bind DNA; the N- terminal portion exhibits no binding activity, suggesting that the C- terminal region contains the DNA binding site. Purified full-length recombinant IN exhibits activity in a specific in vitro assay for enzyme activity involving cleavage of 2 bp from an oligonucleotide corresponding t the HIV-1 LTR. To more precisely localize the DNA binding site within the C terminal half of the molecule, we have constructed a series of additional subclones and analyzed the deleted proteins for DNA binding. This has enabled us to map the DNA binding site to amino acids 180-248. A manuscript is in press in Nucl. Acids Res. and preparation of additional deletions is in progress. We are also collaborating with David Davies at the NIH and providing him with large quantities of IN MAb for generation of Fab fragments for X-ray crystallography of IN, which may eventually facilitate design of enzyme inhibitors.