Conformational Dynamics of HIV Envelope by Single Molecule Spectroscopy

单分子光谱研究 HIV 包膜的构象动力学

• 批准号: 9148226
• 负责人: Krishanu Ray
• 金额: $ 29.55万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9148226
• 关键词: Antibodies; Antiviral Agents; Binding; Biological Assay; CCR5 gene; CD4 Antigens; CXCR4 gene; Cell-Matrix Junction; Characteristics; Chronic; Cleaved cell; Color; Data; Detection; Epitopes; Evaluation; Exhibits; Fab Immunoglobulins; Fluorescence; Fluorescence Anisotropy; Fluorescence Resonance Energy Transfer; Generations; Genetic; HIV; HIV Envelope Protein gp120; HIV Infections; HIV-1; Health; Immunity; Ligand Binding; Measurement; Measures; Mediating; Methods; Molecular Conformation; Nature; Outcome; Phenotype; Positioning Attribute; Reagent; Reporting; Resistance; Series; Spectrum Analysis; Structure; Testing; Time; Vaccines; Viral; Virion; Virus; Virus Replication; base; flexibility; insight; neutralizing antibody; novel; novel strategies; response; single molecule; single-molecule FRET; stoichiometry

 DESCRIPTION (provided by applicant): It is widely held that the establishment and course of HIV infection is determined by the interplay between viral replication and humoral responses to viral envelope trimers that mediate host cell attachment and entry. Accordingly, intensive efforts have been directed towards understanding the structural, functional and antigenic characteristics of HIV trimers. Such information promises to reveal insights for developing antiviral countermeasures, including envelope-targeted antiviral agents and vaccines based on anti-envelope antibodies. The nature of HIV envelope trimers has been examined primarily in the context of free virions. However, several lines of evidence suggest that HIV envelope trimers on virions transition through a series of conformations in solution and/or as they engage up to three CD4 receptor molecules during viral attachment. This in turns suggests certain testable hypotheses concerning the nature of viral trimers. One that will be tested in this project holds that the flexibility and antigenicity of trimers progressively changes as trimers become progressively saturated with CD4. A related hypothesis, also explored in this project, is that certain conformations allow combinations of neutralizing antibodies and/or both neutralizing and non-neutralizing antibodies to bind a single trimer. Our preliminary studies suggest that the latter situation frequently occurs on virions, whereas data from virus capture assays of immunochemical studies with purified soluble trimers suggest that neutralizing epitopes are expressed on "native" trimers in the absence of non-neutralizing epitopes. Accurate evaluations of these hypotheses demands a means to interrogate virions as they naturally exist in solution, without extensive technical manipulation. Previously we reported a single molecule approach based on the use of fluorescence correlation spectroscopy (FCS) to characterize epitope exposures on free virions with all reactants (e.g. antibodies, soluble CD4) continuously in solution. More recently, we have expanded this approach such that we can probe virions simultaneously with multiple antibodies in combination with fluorescence resonance energy transfer (FRET). This novel approach detects multiple epitope exposures on a single virion trimer. It concurrently reveals conformational dynamics as a function of FRET between two epitope probes. Most importantly, such measures are accomplished using our approach without genetic or external perturbations of virions. Thus, we can collect unprecedented information regarding the structural and antigenic dynamics of the HIV envelope, which should provide novel insights for the generation of antiviral agents and anti-HIV immunity. We propose three aims: Aim 1) To characterize the exposure of multiple epitopes on single, virion-associated HIV trimers. Aim 2) To examine the conformational dynamics of gp120 on HIV virions. Aim 3) To characterize conformational flexibility before and during ligand binding to soluble cleaved trimers.

 描述（由适用提供）：被广泛认为，艾滋病毒感染的建立和过程取决于病毒复制和对介导宿主细胞附着和进入的病毒包膜三聚体之间的相互作用和体液响应之间的相互作用。彼此之间，密集型努力是为了理解艾滋病毒三聚体的结构，功能和抗原特征。此类信息有望揭示开发抗病毒对策的见解，包括基于抗炎症抗体的包膜靶向抗病毒剂和疫苗。 HIV信封三聚体的性质主要是在自由病毒的背景下进行的。但是，几条证据表明，通过溶液中的一系列构象和/或，在病毒附着期间，艾滋病毒包膜三聚体通过溶液和/或最多三个CD4受体分子进行过渡。反过来，这表明了有关病毒三聚体性质的某些可检验的假设。将在该项目中进行测试的一种认为，随着三聚体逐渐被CD4饱和，三聚体的柔韧性和抗原性会逐渐变化。在该项目中也探讨了一个相关的假设，是某些会议允许组合中和抗体和/或中和和非中和抗体结合单个三聚体。我们的初步研究表明，后来的情况经常发生在病毒上，而来自病毒的数据捕获了对纯化的固体三聚体进行免疫化学研究的测定，表明在没有非中性表位的情况下，在“天然”三聚体上表达中和表位。对这些假设的准确评估需要一种询问病毒自然存在的方法，而无需进行广泛的技术操纵。以前，我们报告了一种基于荧光相关光谱（FC）的使用来表征与所有反应物（例如抗体，固体CD4）在溶液中连续的自由病毒暴露的表观表位。最近，我们扩展了这种方法，使我们可以简单地探测多种抗体与荧光共振能传递（FRET）探测病毒。这种新颖的方法检测到单个病毒座三聚体上的多发性曝光。它同时揭示了构象动力学，这是两个情节问题之间的FRET的函数。最重要的是，我们提出的三个目标：目标1）表征多个表位对单个与病毒体相关的HIV三聚体上的暴露。目的2）检查HIV包膜的构象动力学，该动力学应该为抗病毒剂和抗HIV免疫学的产生提供新颖的见解。 GP120 HIV病毒。目标3）在配体结合到固化三聚体之前和期间表征概念灵活性。