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）：细胞进入后，逆转录病毒的RNA基因组通过逆转录酶（RT）转换为cDNA拷贝。为了生产性感染，必须将所得病毒cDNA整合到宿主染色体中，这是由整合酶催化的过程（IN）。 HIV-1的某些突变似乎特别损害了逆转录，对生命周期中其他步骤没有明显影响。但是，该缺陷的基本机制知之甚少。在体外，RT和物理相互作用，但是这种RT在相互作用中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在HIV-1复制过程中启动逆转录至关重要。使用核磁共振光谱，我们通过使用基于表面等离子体共振的生物传感器来确定RT相互作用的表面，并确定了RT-IN复合物形成的亲和力和动力学。本应用的目标是更好地了解HIV-1 RT和IN之间的物理相互作用，并确定HIV-1复制过程中RT-IN相互作用的功能意义。 （1）的具体目的是表征HIV-1和RT之间的物理相互作用，并确认RT和在体内发生的相互作用，以及（2）确定HIV-1 RT-IN相互作用的生物学意义。在AIM 1中，我们将使用目标蛋白足迹方法绘制RT的相互作用域。通过使用纯化的病毒体和受感染细胞的细胞质提取物进行共免疫沉淀实验，将确认RT和IN之间的物理相互作用。在AIM 2中，我们将通过破坏假定的RT结合界面并评估病毒复制来测试感染过程中RT-IN相互作用的生物学相关性。我们还将筛选RT突变体，这些突变体可以补偿突变中的RT非相互作用，并检查已知在反转录中产生缺陷的突变体是否具有结合RT的能力。最后，我们将使用基于荧光的高通量屏幕来鉴定RT-IN相互作用的小分子抑制剂，并确定该抑制剂是否还可以阻止病毒复制。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相互作用的表征和确定其生物学意义可能会揭示出新的功能作用，为在突变体中确定的表型提供了机械基础，并确定了抗HIV治疗的新潜在靶标。公共卫生相关性：该提案的目的是了解人类免疫缺陷病毒I型（HIV-1）的两个关键酶，逆转录酶和整合酶之间物理相互作用的重要性。对其生物学意义的相互作用的表征和确定可能揭示了整合酶的新功能，并对病毒的基本生物学有了更深入的了解。此外，该研究可能确定抗HIV治疗的新潜在靶标。