Structure/Function Analysis of the Retrovirus Integrase

逆转录病毒整合酶的结构/功能分析

• 批准号: 7583684
• 负责人: JONATHAN P LEIS
• 金额: $ 22.88万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-12 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7583684
• 关键词: Acids; Acquired Immunodeficiency Syndrome; Affect; Amino Acids; Avian Sarcoma Viruses; Base Pairing; Binding; Binding Sites; Biochemical; Biological; Biology; Cells; Chimera organism; DNA; DNA Binding; DNA Binding Domain; DNA Integration; Defect; Development; Drug Design; Drug resistance; Enzymes; Event; Future; Genome; Growth; HIV-1; Homo; In Vitro; Integrase; Knowledge; Lead; Maps; Modeling; Molecular Genetics; Mutagenesis; Mutation; Naphthyridines; Pharmacotherapy; Positioning Attribute; Principal Investigator; Printing; Process; Property; Reaction; Retroviridae; Site; Site-Directed Mutagenesis; Specificity; Structural Models; Structure; Substrate Interaction; Surface; System; Techniques; Testing; Variant; Viral; Viral Physiology; Virus; base; comparative; crosslink; design; drug development; drug sensitivity; gene therapy; inhibitor/antagonist; innovation; novel; programs; protein complex; public health relevance; reconstitution; site-specific integration; viral DNA

DESCRIPTION (provided by applicant): This is a proposal to carry out a structure/function comparison of two different retrovirus integrases (INs) that highlight both similarities and differences in their respective viral and target DNA binding sites. The study will use a novel and highly predictive homo-tetramer model for HIV-1 IN with bound viral DNA to guide biochemical, biological, and molecular genetic studies. This model identified amino acids in close proximity to the first 15-16 base pairs of the viral DNA ends. These predictions were validated using an innovative strategy that substituted unique amino acid residues from structurally related positions in avian sarcoma virus (ASV) into HIV-1 IN. This produced IN variants that in many cases 3' process heterologous ASV to the detriment of homologous HIV-1 viral LTR DNA end substrates. Three of the variants represent sites where HIV-1 IN amino acid changes occur in the development of diketo acid drug resistance, lead compounds being tested or in use for treatment of AIDS. We now propose to complete mapping of viral and target DNA binding sites and test predictions of the model in the process of integration inside of cells. In Specific Aim 1, we will determine if mutations that disrupt LTR and/or target DNA binding detected using reconstituted systems cause defects to integration of viral into host DNA. For those that do, we will search for 2nd site revertants that restore IN activity and viral growth. We will also demonstrate that the selection of some combinations of amino acids that change in drug resistant HIV-1 integrases is related to affects on 3' processing. Finally, we will identify amino acid residues on ASV IN that recognize its viral DNA ends. This will determine if there is conservation of structure/function across different but functionally related INs. In Specific Aim 2, we will identify amino acids on the IN surface that define the target DNA binding site using mutagenesis, drug sensitivity, and photo-cross linking techniques. Taken together, these innovative studies will provide a new understanding of the mechanism of retrovirus DNA integration and provide a vetted structural model that can be used to examine IN/host protein complexes, for drug design, and future development of enzymes capable of site specific integration. PUBLIC HEALTH RELEVANCE: These innovative studies will add significantly to our knowledge of the mechanism of retrovirus catalyzed DNA integration. They impact on the design of inhibitors directed towards IN and identify amino acid residues likely to be substituted in drug resistant enzymes and why.

描述（由申请人提供）：这是一项对两种不同的逆转录病毒整合酶（IN）进行结构/功能比较的提案，突出了它们各自的病毒和靶DNA结合位点的相似性和差异。该研究将使用一种新颖且高度预测的 HIV-1 IN 与病毒 DNA 结合的同源四聚体模型来指导生化、生物学和分子遗传学研究。该模型识别出紧邻病毒 DNA 末端前 15-16 个碱基对的氨基酸。这些预测通过一种创新策略得到验证，该策略将禽肉瘤病毒 (ASV) 结构相关位置的独特氨基酸残基替换为 HIV-1 IN。这产生了 IN 变体，在许多情况下，这些变体对异源 ASV 进行 3' 加工，从而损害同源 HIV-1 病毒 LTR DNA 末端底物。其中三个变体代表在二酮酸耐药性发展过程中发生 HIV-1 IN 氨基酸变化的位点，正在测试或用于治疗艾滋病的先导化合物。我们现在建议完成病毒和靶DNA结合位点的绘制，并测试模型在细胞内整合过程中的预测。在具体目标 1 中，我们将确定使用重组系统检测到的破坏 LTR 和/或目标 DNA 结合的突变是否会导致病毒整合到宿主 DNA 中的缺陷。对于那些这样做的人，我们将寻找能够恢复 IN 活性和病毒生长的第二位点回复体。我们还将证明，在耐药性 HIV-1 整合酶中发生变化的某些氨基酸组合的选择与对 3' 加工的影响有关。最后，我们将鉴定 ASV IN 上识别其病毒 DNA 末端的氨基酸残基。这将确定不同但功能相关的 IN 之间是否存在结构/功能保守。在具体目标 2 中，我们将使用诱变、药物敏感性和光交联技术来识别 IN 表面上定义目标 DNA 结合位点的氨基酸。总而言之，这些创新研究将为逆转录病毒 DNA 整合机制提供新的认识，并提供经过审查的结构模型，可用于检查 IN/宿主蛋白复合物、药物设计以及能够进行位点特异性整合的酶的未来开发。公共卫生相关性：这些创新研究将显着增加我们对逆转录病毒催化 DNA 整合机制的了解。它们影响针对 IN 的抑制剂的设计，并确定耐药酶中可能被取代的氨基酸残基及其原因。