HIV-1 INTEGRATION PROTEIN

HIV-1 整合蛋白

• 批准号: 2066282
• 负责人: DUANE P GRANDGENETT
• 金额: $ 17.14万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 1996-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2066282
• 关键词: DNA binding protein; Escherichia coli; analytical ultracentrifugation; antisense nucleic acid; circular dichroism; endonuclease; genetic translation; host organism interaction; human immunodeficiency virus 1; microorganism genetics; nucleic acid repetitive sequence; protein purification; protein sequence; recombinant DNA; transfection; virus protein

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.

逆转录病毒的整合蛋白（IN）是整合所必需的