HIV-1 INTEGRATION PROTEIN

逆转录病毒的整合蛋白（IN）是整合所必需的 线性病毒DNA插入宿主DNA 线性合成后 平端DNA，IN从 两者的3’-OH末端 病毒链 随后通过IN将3'凹进端附接至 在没有外源能量的情况下，宿主DNA的5 '-磷酰基末端 源头 该赠款提案的重点是HIV-1 IN参与 两个融合步骤。 HIV-1 IN已成功表达于 大肠杆菌和部分纯化。 纯化的IN具有 对HIV-1末端具有特异性3 ′-OH末端核酸内切酶活性， 能够将HIV-1 LTR寡核苷酸插入另一个DNA 分子。 目前正在努力将IN纯化到明显的 同质性 溶液化学包括圆二色性， IN的二级结构和分析离心，以确定 沉降系数和IN的分子量。 一个 用于测量和定量HIV-1 3 '-OH末端的酸溶性测定 正在开发核酸内切酶活性。 一种快速灵敏的检测方法将 对潜在抑制剂的开发和定量很重要 HIV-1 IN核酸内切酶活性的增加，这对于第一步 一体化 体外整合重组检测方法的建立 使用纯化的IN将提供试剂来定义涉及的机制， 整合以及鉴定第二步的抑制剂， 整合，通过IN将3'凹陷末端插入DNA中。 努力 将扩展到使用温和的 在存在和不存在特异性HIV-1 LTR的情况下IN的蛋白酶消化 寡核苷酸 初步研究表明，HIV-1 病毒体中的IN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水解处理，将完成。 错义突变将被引入IN基因的特定区域 来识别非致命突变。 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.