Role of HIV-1 IN during reverse transcription and uncoating

HIV-1 IN 在逆转录和脱壳过程中的作用

• 批准号: 8139493
• 负责人: Samson A Chow
• 金额: $ 28.76万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8139493
• 关键词: Adopted; Affect; Affinity; Amino Acids; Anti-HIV Therapy; Binding; Biological; Biological Testing; Biology; Biosensor; C-terminal; Capsid; Cells; Chromosomes; Complementary DNA; Complex; Cyclophilin A; DNA biosynthesis; Data; Defect; Defective Viruses; Deletion Mutation; Enzymes; Fluorescence; Genome; Goals; HIV; HIV-1; Human; In Vitro; Infection; Integrase; Integration Host Factors; Kinetics; Life Cycle Stages; Light; Macaca mulatta; Maps; Methods; Molecular Conformation; Mutate; Mutation; NMR Spectroscopy; Nuclear Import; Nuclear Magnetic Resonance; Optics; Peptide Hydrolases; Peptidylprolyl Isomerase; Post-Translational Protein Processing; Process; Protein Footprinting; Proteins; RNA; RNA-Directed DNA Polymerase; Recombinant Proteins; Recombinants; Reverse Transcriptase Inhibitors; Reverse Transcription; Role; Site; Surface; Surface Plasmon Resonance; Viral; Viral Genome; Viral Reverse Transcription; Virus; base; ds-DNA; high throughput screening; in vivo; inhibitor/antagonist; mutant; small molecule; viral DNA

DESCRIPTION (provided by applicant): Retroviral integrase (IN) catalyzes the essential step of integrating the double-stranded DNA copy of the viral genome into the chromosome of an infected cell. Certain mutations of HIV-1 IN can 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, HIV-1 IN physically interacts with reverse transcriptase (RT) and stimulates reverse transcription, but the biological relevance of this RT-IN interaction is not known. 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. In addition to RT interaction, we recently have characterized HIV-1 IN mutations that resulted in poor core yield and instability, and reduced incorporation of cyclophilin A (CypA), a cellular peptidyl-prolyl isomerase that binds specifically to capsid (CA). Further analyses indicate that IN is required during uncoating by maintaining CypA-CA interaction, which promotes optimal stability of the viral core. Taken together, we hypothesize that HIV-1 IN can affect reverse transcription either directly by interacting with RT or indirectly by altering uncoating. The goal of this application is to gain a better understanding of the functional role of IN during uncoating and reverse transcription. The specific aims are (1) to determine the biological significance of the HIV-1 RT-IN interaction, (2) to characterize the physical interaction between HIV-1 RT and IN, and examine other factors that can modulate the RT-IN interaction, and (3) to understand the mechanism by which IN affects the uncoating of viral cores. In Aim 1, we will test the biological relevance of the RT-IN interaction during infection by disrupting the putative IN-RT binding interface and assessing the impact on reverse transcription and viral replication. We will also screen for RT mutants that can compensate for the RT- noninteracting IN mutations, and use a fluorescence-based high-throughput screen to identify small-molecule inhibitors of the RT-IN interaction and determine whether such inhibitors also block viral replication. In Aim 2, we will map the IN-interacting domain of RT using a targeted protein footprinting method. We will also examine the effect of a host factor SIP1, which binds specifically to IN and is required for reverse transcription, on the RT-IN binding interface, and determine if SIP1 can affect IN's activities on nuclear import and integration. In Aim 3, we will determine if the IN-CA interaction in the viral core requires post-translational modification or a bridge factor. We will also examine the effect of CypA- and TRIM51-binding to CA on IN's role during uncoating, and the relationship between uncoating and reverse transcription. In the process, we will shed light on the interactions among key retroviral proteins and their host factors, and the effect of such interactions on reverse transcription and uncoating. Characterization of the interactions and determination of their biological significance may reveal new functional roles for IN, and identify new potential targets for anti-HIV therapy. PUBLIC HEALTH RELEVANCE: The objective of this proposal is to understand the functional role of integrase (IN), a key enzyme of human immunodeficiency virus type I (HIV-1), during the uncoating and reverse transcription steps of the viral life cycle. We will study the interaction between IN and another key enzyme, reverse transcriptase, and the effect of the interaction on reverse transcription. We will also determine the mechanism by which IN affects capsid disassembly during uncoating. Characterization of the interactions and determination of their biological significance may reveal new functional roles for IN 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）：逆转录病毒整合酶（IN）催化将病毒基因组的双链DNA拷贝整合到受感染细胞的染色体中的重要步骤。 HIV-1 IN 的某些突变可以特异性损害逆转录，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，HIV-1 IN 与逆转录酶 (RT) 发生物理相互作用并刺激逆转录，但这种 RT-IN 相互作用的生物学相关性尚不清楚。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。除了 RT 相互作用之外，我们最近还描述了 HIV-1 IN 突变，这些突变导致核心产量差和不稳定，并减少亲环蛋白 A (CypA) 的掺入，亲环蛋白 A (CypA) 是一种与衣壳 (CA) 特异性结合的细胞肽基脯氨酰异构酶。进一步的分析表明，在脱壳过程中需要 IN 来维持 CypA-CA 相互作用，从而促进病毒核心的最佳稳定性。综上所述，我们假设 HIV-1 IN 可以通过与 RT 相互作用直接影响逆转录，或者通过改变脱壳间接影响逆转录。本应用的目的是更好地了解 IN 在脱壳和逆转录过程中的功能作用。具体目标是 (1) 确定 HIV-1 RT-IN 相互作用的生物学意义，(2) 表征 HIV-1 RT 和 IN 之间的物理相互作用，并检查可以调节 RT-IN 相互作用的其他因素，（3）了解IN影响病毒核心脱壳的机制。在目标 1 中，我们将通过破坏假定的 IN-RT 结合界面并评估对逆转录和病毒复制的影响来测试感染期间 RT-IN 相互作用的生物学相关性。我们还将筛选可以补偿 RT-非相互作用 IN 突变的 RT 突变体，并使用基于荧光的高通量筛选来识别 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。在目标 2 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。我们还将检查宿主因子SIP1（它与IN特异性结合并且是逆转录所需的）对RT-IN结合界面的影响，并确定SIP1是否可以影响IN在核输入和整合方面的活性。在目标 3 中，我们将确定病毒核心中的 IN-CA 相互作用是否需要翻译后修饰或桥接因子。我们还将研究 CypA 和 TRIM51 与 CA 结合对 IN 在脱衣过程中的作用的影响，以及脱衣和逆转录之间的关系。在此过程中，我们将阐明关键逆转录病毒蛋白与其宿主因子之间的相互作用，以及这种相互作用对逆转录和脱壳的影响。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，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整合酶 (IN) 在病毒生命周期的脱壳和逆转录步骤中的功能作用，整合酶是 I 型人类免疫缺陷病毒 (HIV-1) 的关键酶。我们将研究IN与另一种关键酶逆转录酶之间的相互作用，以及相互作用对逆转录的影响。我们还将确定 IN 在脱衣过程中影响衣壳分解的机制。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，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。