Structure/Function Analysis of the Retrovirus Integrase

描述（由申请方提供）：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，突出了其各自病毒和靶DNA结合位点的相似性和差异。该研究将使用一种新的和高度预测性的HIV-1 IN与病毒DNA结合的同源四聚体模型来指导生物化学，生物学和分子遗传学研究。该模型鉴定了与病毒DNA末端的前15-16个碱基对非常接近的氨基酸。这些预测得到了验证，使用一种创新的策略，取代独特的氨基酸残基从禽肉瘤病毒（ASV）到HIV-1 IN的结构相关的位置。这产生了IN变体，其在许多情况下3'加工异源ASV而损害同源HIV-1病毒LTR DNA末端底物。其中三种变异体代表了HIV-1 IN氨基酸在二酮酸耐药性发展中发生变化的位点，这些位点是正在测试或用于治疗AIDS的先导化合物。我们现在建议完成病毒和靶DNA结合位点的映射，并在细胞内整合的过程中测试模型的预测。在特定目标1中，我们将确定使用重建系统检测到的破坏LTR和/或靶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，我们将寻找第二个网站回复突变体，恢复IN的活动和病毒的增长。我们还将证明，在耐药HIV-1整合酶中改变的一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。这将确定是否有不同的，但功能相关的INS结构/功能的保护。在具体目标2中，我们将确定使用诱变，药物敏感性，和光交联技术的IN表面上的氨基酸定义的目标DNA结合位点。总之，这些创新的研究将提供一个新的理解逆转录病毒DNA整合的机制，并提供了一个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，以及未来的开发酶能够位点特异性整合。公共卫生相关性：这些创新的研究将大大增加我们对逆转录病毒催化的DNA整合机制的了解。它们影响针对IN的抑制剂的设计，并确定可能在耐药酶中被取代的氨基酸残基及其原因。

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.