HIV-1 INTEGRATION PROTEIN

逆转录病毒的整合蛋白（IN）是整合所必需的 将线性病毒DNA转化为宿主DNA。 以下合成线性 平端 DNA，IN 从两者的 3'-OH 末端去除 2 个碱基 病毒链。 3' 凹进端随后通过 IN 连接到 没有外源能量时宿主 DNA 的 5'-磷酰基末端 来源。 赠款提案的重点是 HIV-1 IN 参与 两个集成步骤。 HIV-1 IN已成功表达于 大肠杆菌并部分纯化。 Purified IN 具有 HIV-1 末端的特异性 3'-OH 末端核酸内切酶活性是 能够将 HIV-1 LTR 寡核苷酸插入另一个 DNA 中 分子。 目前正在努力将 IN 纯化为表观 同质性。 溶液化学包括圆二色性来定义 IN 的二级结构和分析离心来定义 将启动IN的沉降系数和分子量。 安 用于测量和定量 HIV-1 3'-OH 末端的酸溶测定法 核酸内切酶活性正在开发中。 快速、灵敏的检测将 对于潜在抑制剂的开发和定量很重要 HIV-1 IN 核酸内切酶活性，这对于第一步至关重要 整合。 体外整合重组测定的开发 使用纯化的 IN 将提供试剂来定义所涉及的机制 整合以及识别第二步的抑制剂 整合，通过 IN 将 3' 凹进末端插入 DNA。 努力 将使用温和的方法扩展以绘制 HIV-1 IN 的 DNA 结合域图谱 在存在或不存在特定 HIV-1 LTR 的情况下对 IN 进行蛋白酶消化 寡核苷酸。 初步研究表明，HIV-1 病毒体中的 IN 是非磷酸化的单一多肽（288 个残基），不 在其羧基末端进行蛋白水解加工，将完成。 错义突变将被引入 IN 基因的特定区域 识别非致死突变。 这些遗传学研究可能有助于 绘制 IN 的酶结构域。

The integration protein (IN) of retroviruses is necessary for integration of linear viral DNA into host DNA. Following synthesis of linear blunt-ended DNA, IN removes 2 bases from the 3'-OH termini of both viral strands. The 3' recessed ends are subsequently attached by IN to the 5'-phosphoryl ends of the host DNA in the absence of an exogenous energy source. The grant proposal focuses on the participation of HIV-1 IN in both of the integration steps. HIV-1 IN has been successfully expressed in Escherichia coli and partially purified. Purified IN possesses both a specific 3'-OH terminal endonuclease activity for HIV-1 termini and is capable of inserting an HIV-1 LTR oligonucleotide into another DNA molecule. Efforts are currently underway to purify IN to apparent homogeneity. Solution chemistry including circular dichroism to define secondary structure of IN and analytical centrifugation to define the sedimentation coefficient and molecular weight of IN will be initiated. An acid-soluble assay for measuring and quantitating the HIV-1 3'-OH terminal endonuclease activity is being developed. A rapid and sensitive assay will be important for the development and quantitation of potential inhibitors of HIV-1 IN endonuclease activity which is essential for the first step of integration. Development of an in vitro integrative recombination assay using purified IN will provide reagents to define the mechanism involved in integration as well as identifying inhibitors of the second step of integration, the insertion of 3' recessed ends into DNA by IN. Efforts will be extended to map the DNA binding domain of HIV-1 IN using mild protease digestion of IN in the presence and absence of specific HIV-1 LTR oligonucleotides. Preliminary studies, which have demonstrated that HIV-1 IN in virions is a non-phosphorylated single polypeptide (288 residues) not proteolytically processed at its carboxylterminus, will be completed. Missense mutations will be introduced into specific regions of the IN gene to identify non-lethal mutations. These genetic studies may be useful for mapping the enzymatic domain(s) of IN.