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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

基本信息

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
项目类别：
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

项目摘要

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（由申请方提供）：逆转录病毒整合酶（IN）催化将病毒基因组的双链DNA拷贝整合到感染细胞染色体中的重要步骤。HIV-1 IN的某些突变可以特异性地损害逆转录，而对生命周期中的其他步骤没有明显影响。然而，这种缺陷的潜在机制知之甚少。在体外，HIV-1 IN与逆转录酶（RT）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。使用核磁共振光谱，我们已经确定了RT相互作用的表面上IN，并确定了RT-IN复合物形成的亲和力和动力学，通过使用表面等离子体共振为基础的生物传感器。除了RT相互作用，我们最近已经表征了HIV-1 IN突变，其导致较差的核心产量和不稳定性，以及亲环蛋白A（CypA）（一种特异性结合衣壳（CA）的细胞肽基脯氨酰异构酶）的掺入减少。进一步的分析表明，通过维持CypA-CA相互作用，在脱壳期间需要IN，这促进了病毒核心的最佳稳定性。综上所述，我们假设HIV-1 IN可以直接通过与RT相互作用或间接通过改变未包被来影响逆转录。本申请的目的是更好地了解IN在脱膜和逆转录过程中的功能作用。具体的目的是（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，以及（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阻断病毒复制。在目标2中，我们将使用靶向蛋白足迹法绘制RT的IN相互作用结构域。我们还将研究一个主机因子SIP 1，它特异性地结合到IN和逆转录所需的，在RT-IN结合界面的效果，并确定SIP 1是否可以影响IN的活动核进口和整合。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因子。我们还将研究CypA-和TRIM 51-结合CA对IN在脱壳过程中的作用的影响，以及脱壳和逆转录之间的关系。在这个过程中，我们将阐明关键的逆转录病毒蛋白和它们的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。表征的相互作用和确定其生物学意义可能揭示新的功能作用的IN，并确定新的潜在的抗HIV治疗的目标。公共卫生相关性：本提案的目的是了解整合酶（IN），人类免疫缺陷病毒I型（HIV-1）的关键酶，在病毒生命周期的脱壳和逆转录步骤的功能作用。我们将研究IN与另一个关键酶逆转录酶的相互作用，以及这种相互作用对逆转录的影响。我们还将确定在脱壳过程中IN影响衣壳解体的机制。表征的相互作用和确定其生物学意义可能会揭示新的功能作用的IN和提供更深入的了解病毒的基本生物学。此外，该研究可能会发现抗HIV治疗的新的潜在靶点。