Characterizing the binding between HIV-1 integrase and capsid and the role of the interaction in modulating the uncoating process

表征 HIV-1 整合酶和衣壳之间的结合以及相互作用在调节脱衣过程中的作用

• 批准号: 8924724
• 负责人: Samson A Chow
• 金额: $ 18.43万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-08 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8924724
• 关键词: Adopted; Affect; Amino Acids; Anti-HIV Therapy; Area; Binding; Biochemical; Biological; Biological Assay; Biology; Capsid; Capsid Proteins; Cell membrane; Cells; Chromosomes; Complement; Complex; Core Assembly; Cryoelectron Microscopy; Cyclophilin A; Cytoplasm; Data; Defect; Encapsulated; Enzymes; Event; Genome; Goals; HIV; HIV-1; Impairment; In Vitro; Individual; Infection; Integrase; Integrase Inhibitors; Life Cycle Stages; Light; Maps; Measures; Molecular Conformation; Molecular Sieve Chromatography; Morphology; Mutation; Peptidylprolyl Isomerase; Phase; Phenotype; Process; Proteins; Retroviridae; Reverse Transcription; Ribonucleoproteins; Role; Shapes; Specificity; Staging; Structural Protein; Structure; Surface; Surface Plasmon Resonance; Tubular formation; Viral; Viral Genome; Viral Proteins; Virion; Virus; detector; ds-DNA; electron tomography; insight; light scattering; mutant; particle; public health relevance; stoichiometry; three dimensional structure; viral RNA

 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. However, certain mutations of HIV-1 IN can impair reverse transcription, uncoating, and core morphology, and the underlying mechanisms for these defects are not well understood. We have characterized HIV-1 IN mutations that resulted in non-infectious viruses with low core yield and poor stability, and impairment at the step of early reverse transcription. Further analyses indicate that IN enhances core incorporation of cyclophilin A (CypA), a cellular peptidyl-prolyl isomerase that binds specifically to capsid (CA) and promotes optimal stability of the viral core. We hypothesize that HIV-1 IN can impact uncoating by interacting with CA, which in turn affects the incorporation of CypA to the core. We have preliminary data indicating that IN interacts with CA within HIV-1 cores. We have further found that purified IN does not bind monomeric or dimeric CA, but can readily bind in vitro assembled core-like structures formed with purified CA or CA-NC, as well as a CA mutant that preferentially forms hexamers under non-reducing conditions. The goal of this application is to gain a better understanding of the interaction between HIV-1 IN and CA and the functional role of the IN-CA interaction during HIV-1 replication. The specific aims are (1) to characterize the physical interaction between HIV-1 IN and higher-order CA structures, and (2) to visualize the morphology of CA-noninteracting IN mutant viruses and the structures of HIV-1 IN bound with CA assemblies and CA hexamers. In Aim 1, we will use CA hexamers and core-like structures formed with purified CA as substrates in binding assays to determine the binding specificity and map the key domains and particular amino acids on IN involved in binding. To further confirm the IN-CA assembly interaction and to determine the oligomeric states preferred for binding and the stoichiometry and binding constants, we will use size exclusion chromatography-multi angle light scattering detector (SEC-MALS) and surface plasmon resonance (SPR) to measure IN-CA binding. We will also investigate the role of the IN-CA interaction at the core assembly step during the late phase of HIV-1 life cycle. In Aim 2, we will obtain three dimensional structures of the CA-noninteracting IN mutant viruses by cryo-electron tomography (cryo-ET), and use cryo-electron microscopy (cryo-EM) to analyze CA assemblies or CA hexamers bound with IN as another approach to examine the IN-CA interaction. The cryo-EM study may also reveal the IN binding surface on the CA multimers, which will corroborate the results obtained from biochemical and other biophysical analyses. In the process, we will shed light on the interaction between IN and CA, and the effect of such interaction on 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.

 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。然而，HIV-1 IN的某些突变会损害逆转录，我们对 HIV-1 IN 突变的特征还不清楚，这些突变导致非感染性病毒核心产量低、稳定性差，并且在步骤中受到损害。进一步的分析表明，IN 增强了亲环蛋白 A (CypA) 的核心掺入，这是一种与衣壳 (CA) 特异性结合的细胞肽基脯氨酰异构酶，并促进病毒核心的最佳稳定性。可以通过与 CA 相互作用来影响脱壳，这反过来又影响 CypA 与核心的结合。我们有初步数据表明 IN 与 HIV-1 核心内的 CA 相互作用。纯化的IN不结合单体或二聚体CA，但可以容易地结合由纯化的CA或CA-NC形成的体外组装的核心样结构，以及在非还原条件下优先形成六聚体的CA突变体。应用的目的是更好地了解 HIV-1 IN 和 CA 之间的相互作用以及 IN-CA 相互作用在 HIV-1 复制过程中的功能作用。具体目标是 (1) 表征 HIV-1 IN 之间的物理相互作用。和高阶 CA 结构，以及(2) 可视化与 CA 不相互作用的 IN 突变病毒的形态以及与 CA 组装体和 CA 六聚体结合的 HIV-1 IN 的结构。在目标 1 中，我们将使用以纯化的 CA 形成的 CA 六聚体和核心状结构作为底物。用于确定结合特异性并绘制 IN 上参与结合的关键结构域和特定氨基酸的图谱，以进一步确认 IN-CA 组装相互作用并确定首选结合的寡聚状态和化学计量。结合常数，我们将使用尺寸排阻色谱-多角度光散射检测器 (SEC-MALS) 和表面等离子共振 (SPR) 来测量 IN-CA 结合，我们还将研究 IN-CA 相互作用在核心组件中的作用。在目标2中，我们将通过冷冻电子断层扫描（cryo-ET）获得CA非相互作用IN突变病毒的三维结构，并使用冷冻电子。冷冻电镜研究还可以揭示 CA 多聚体上的 IN 结合表面，这将证实所获得的结果。在此过程中，我们将阐明 IN 和 CA 之间的相互作用，以及这种相互作用对脱壳的影响，并确定其生物学意义可能会揭示新的功能作用。对于 IN，并确定 抗艾滋病毒治疗的新潜在目标。