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(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基本步骤。然而，HIV-1IN的某些突变可以损害反转录、去涂层和核心形态，并且这些缺陷的潜在机制还不清楚。我们已经确定了HIV-1IN突变的特征，这些突变导致非传染性病毒具有低核心产量和较差的稳定性，并在早期逆转录阶段受损。进一步的分析表明，IN增强了亲环素A(CypA)的核心掺入，CypA是一种细胞内肽-Pro异构酶，与衣壳(CA)特异结合，促进病毒核心的最佳稳定性。我们假设HIV-1 IN可以通过与CA相互作用影响去涂层，这反过来又影响CypA到核心的掺入。我们有初步数据表明，IN与HIV-1核心中的CA相互作用。我们进一步发现，纯化的IN不结合单体或二聚体CA，但在体外可以很容易地结合由纯化的CA或CA-NC形成的核心状结构，以及在非还原条件下优先形成六聚体的CA突变体。本应用的目的是为了更好地了解HIV-1IN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。其具体目的是(1)表征HIV-1 IN与高阶CA结构之间的物理相互作用，(2)可视化CA非相互作用IN突变病毒的形态以及HIV-1 IN与CA组件和CA六聚体结合的结构。在目标1中，我们将使用CA六聚体和由纯化的CA形成的核样结构作为结合分析的底物来确定结合特异性，并将参与结合的关键结构域和特定氨基酸定位在IN上。为了进一步证实IN-CA组装相互作用，并确定首选结合的低聚态以及化学计量比和结合常数，我们将使用尺寸排除色谱-多角度光散射检测器(SEC-MALS)和表面等离子体共振(SPR)来测量IN-CA结合。我们还将研究IN-CA相互作用在HIV-1生命周期后期核心组装步骤中的作用。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，并使用冷冻电子显微镜(Cryo-EM)分析CA组装或与IN结合的CA六聚体，作为另一种检查IN-CA相互作用的方法。冷冻-EM研究还可能揭示CA多聚体上的IN结合表面，这将证实生化和其他生物物理分析的结果。在这个过程中，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涂层的影响。相互作用的特征和生物学意义的确定可能揭示IN的新功能角色，并识别 抗艾滋病毒治疗的新潜在靶点。