HIV-1 INTEGRATION PROTEIN

逆转录病毒的整合蛋白(IN)是整合所必需的 将线状病毒DNA转化为宿主DNA。在线性合成之后 钝端DNA，IN从两者的3‘-OH末端去除2个碱基 病毒链。3‘凹端随后通过IN连接到 在没有外源能量的情况下宿主DNA的5‘-磷酸末端 消息来源。赠款提案的重点是艾滋病毒-1参与在 这两个集成步骤。HIV-1 IN已成功地在 大肠埃希氏菌，部分纯化。PURIZED IN同时拥有 HIV-1末端和IS的特异性3‘-OH末端内切酶活性 能够将HIV-1LTR寡核苷酸插入到另一DNA中 分子。目前正在努力净化表面上的 同质性。包括圆二色谱在内的溶液化学定义 IN的二级结构和分析离心法确定 将启动IN的沉降系数和分子量。一个 测定和定量HIV-1 3‘-OH末端的酸溶法 核酸内切酶活性正在开发中。一种快速而灵敏的分析方法将 对潜在抑制剂的开发和定量具有重要意义 HIV-1在核酸内切酶活性中的作用，这对第一步至关重要 整合。一种体外整合重组实验方法的建立 使用纯化的IN将提供试剂来确定参与的机制 整合以及确定第二步的抑制因素 整合，通过IN将3‘端凹陷末端插入到DNA中。努力 将被扩展到绘制HIV-1的DNA结合域 在存在和不存在HIV-1特异性LTR的情况下IN的蛋白酶消化 寡核苷酸。初步研究表明，艾滋病毒-1 In在病毒粒子中是一种非磷酸化的单一多肽(288个残基) 在其羧基末端进行蛋白质水解性加工，将完成。 错义突变将被引入IN基因的特定区域 以确定非致命性突变。这些遗传学研究可能对 定位IN的酶切域(S)。

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.