Binding Between HIV-1 RT and IN and Its Functional Signification

HIV-1 RT与IN的结合及其功能意义

• 批准号: 7555091
• 负责人: Samson A Chow
• 金额: $ 17.97万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-10 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7555091
• 关键词: Affinity; Amino Acids; Anti-HIV Therapy; Binding; Biological; Biological Assay; Biological Testing; Biology; Biosensor; C-terminal; Cells; Chimeric Proteins; Chromosomes; Co-Immunoprecipitations; Complementary DNA; Complex; Data; Defect; Defective Viruses; Deletion Mutation; Depth; Enzymes; Exhibits; Fluorescence; Genetic Transcription; Genome; Goals; HIV; HIV-1; In Vitro; Infection; Integrase; Kinetics; Life Cycle Stages; Light; Maps; Methods; Mutate; Mutation; NMR Spectroscopy; Nuclear Magnetic Resonance; Optics; Phenotype; Play; Process; Protein Footprinting; Public Health; Publishing; RNA; RNA-Directed DNA Polymerase; Rate; Recombinant Proteins; Recombinants; Reporting; Retroviridae; Reverse Transcriptase Inhibitors; Reverse Transcription; Role; Surface; Surface Plasmon Resonance; Viral; Viral Genome; Viral Reverse Transcription; Virion; Virus; base; ds-DNA; high throughput screening; in vivo; inhibitor/antagonist; mutant; research study; small molecule

DESCRIPTION (provided by applicant): After cell entry, the RNA genome of retroviruses is converted to a cDNA copy by reverse transcriptase (RT). For productive infection, the resulting viral cDNA must be integrated into the host chromosome, a process catalyzed by integrase (IN). Certain mutations of HIV-1 IN appear to specifically impair reverse transcription with no apparent effects on other steps in the life cycle. However, the underlying mechanism for this defect is poorly understood. In vitro, RT and IN physically interact, but the biological relevance of this RT- IN interaction is not known. We hypothesize that the physical interaction between RT and IN is functional and critical for initiating reverse transcription during HIV-1 replication. Using nuclear magnetic resonance spectroscopy, we have identified the RT-interacting surface on IN, and have determined the affinity and kinetics of RT-IN complex formation by using a surface plasmon resonance-based biosensor. The goals of this application are to gain a better understanding of the physical interaction between HIV-1 RT and IN, and to determine the functional significance of the RT-IN interaction during HIV-1 replication. The specific aims are (1) to characterize the physical interaction between HIV-1 IN and RT and confirm that the interaction between RT and IN occurs in vivo, and (2) to determine the biological significance of the HIV-1 RT-IN interaction. In Aim 1, we will map the IN-interacting domain of RT using a targeted protein footprinting method. The physical interaction between RT and IN during replication will be confirmed by carrying out a co-immunoprecipitation experiment using purified virions and cytoplasmic extracts from infected cells. In Aim 2, we will test the biological relevance of the RT-IN interaction during infection by disrupting the putative IN-RT binding interface and assessing viral replication. We will also screen for RT mutants that can compensate for the RT- noninteracting IN mutations and examine other IN mutants known to produce defects in reverse transcription for their ability to bind RT. Finally, we will identify small-molecule inhibitors of the RT-IN interaction using a fluorescence-based high-throughput screen and determine whether such inhibitors also block viral replication. In the process, we will shed light on the interaction between two key retroviral enzymes and the effect of such an interaction on reverse transcription and viral replication. Characterization of the RT-IN interaction and determination of its biological significance may reveal new functional roles for IN, provide a mechanistic basis for the phenotypes observed with certain IN mutants, and identify new potential targets for anti-HIV therapy. PUBLIC HEALTH RELEVANCE: The objective of this proposal is to understand the importance of the physical interaction between two key enzymes, reverse transcriptase and integrase, of human immunodeficiency virus type I (HIV-1). Characterization of the interaction and determination of its biological significance may reveal new functional roles for integrase and provide a deeper understanding of the basic biology of the virus. Furthermore, the study may identify new potential targets for anti-HIV therapy.

描述（由申请人提供）：进入细胞后，逆转录病毒的RNA基因组通过逆转录酶（RT）转化为cDNA拷贝。为了有效感染，产生的病毒 cDNA 必须整合到宿主染色体中，这一过程由整合酶 (IN) 催化。 HIV-1 IN 的某些突变似乎会特异性损害逆转录，而对生命周期的其他步骤没有明显影响。然而，人们对该缺陷的潜在机制知之甚少。在体外，RT 和 IN 发生物理相互作用，但这种 RT-IN 相互作用的生物学相关性尚不清楚。我们假设 RT 和 IN 之间的物理相互作用对于 HIV-1 复制期间启动逆转录具有功能性且至关重要。使用核磁共振波谱，我们识别了 IN 上的 RT 相互作用表面，并通过使用基于表面等离子体共振的生物传感器确定了 RT-IN 复合物形成的亲和力和动力学。本应用的目的是更好地了解 HIV-1 RT 和 IN 之间的物理相互作用，并确定 RT-IN 相互作用在 HIV-1 复制过程中的功能意义。具体目标是 (1) 表征 HIV-1 IN 和 RT 之间的物理相互作用，并确认 RT 和 IN 之间的相互作用发生在体内，以及 (2) 确定 HIV-1 RT-IN 相互作用的生物学意义。在目标 1 中，我们将使用靶向蛋白质足迹方法绘制 RT 的 IN 相互作用域图。 RT 和 IN 在复制过程中的物理相互作用将通过使用纯化的病毒粒子和感染细胞的细胞质提取物进行免疫共沉淀实验来证实。在目标 2 中，我们将通过破坏假定的 IN-RT 结合界面并评估病毒复制来测试感染期间 RT-IN 相互作用的生物学相关性。我们还将筛选能够补偿与 RT 不相互作用的 IN 突变的 RT 突变体，并检查已知会在逆转录中产生缺陷的其他 IN 突变体的结合 RT 的能力。最后，我们将使用基于荧光的高通量筛选来鉴定 RT-IN 相互作用的小分子抑制剂，并确定此类抑制剂是否也能阻止病毒复制。在此过程中，我们将阐明两种关键逆转录病毒酶之间的相互作用以及这种相互作用对逆转录和病毒复制的影响。 RT-IN 相互作用的表征及其生物学意义的确定可能会揭示 IN 新的功能作用，为某些 IN 突变体观察到的表型提供机制基础，并确定抗 HIV 治疗的新潜在靶点。公共健康相关性：本提案的目的是了解人类免疫缺陷病毒 I 型 (HIV-1) 的两种关键酶（逆转录酶和整合酶）之间物理相互作用的重要性。相互作用的表征及其生物学意义的确定可能会揭示整合酶的新功能作用，并提供对病毒基础生物学的更深入的了解。此外，该研究可能会确定抗艾滋病毒治疗的新潜在靶点。