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: 9340245
• 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/9340245
• 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; Fluorescence Spectroscopy; Generations; Genetic; HIV; HIV Envelope Protein gp120; HIV Infections; HIV-1; 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; public health relevance; 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（由申请人提供）：人们普遍认为，HIV 感染的建立和过程是由病毒复制和对病毒包膜三聚体的体液反应之间的相互作用决定的，病毒包膜三聚体介导宿主细胞附着和进入，了解其结构、功能和抗原特征。这些信息有望揭示开发抗病毒对策的见解，包括针对包膜的抗病毒药物和基于抗包膜抗体的疫苗。 HIV 包膜三聚体的性质主要在游离病毒粒子的背景下进行了研究。然而，一些证据表明，病毒粒子上的 HIV 包膜三聚体在溶液中和/或当它们与多达三个 CD4 受体分子结合时通过一系列构象转变。这反过来表明了有关病毒三聚体性质的某些可测试假设，即随着三聚体逐渐被 CD4 饱和，三聚体的灵活性和抗原性也会逐渐变化。在这个项目中探索的是，某些构象允许中和抗体和/或中和抗体和非中和抗体结合单个三聚体，我们的初步研究表明后一种情况经常发生在病毒体上，而来自病毒捕获测定的数据。使用纯化的可溶性三聚体进行的免疫化学研究表明，在不存在非中和表位的情况下，中和表位在“天然”三聚体上表达。对这些假设的准确评估需要一种询问病毒体的方法。因为它们自然存在于溶液中，无需进行大量的技术操作，之前我们报道了一种基于荧光相关光谱 (FCS) 的单分子方法，以连续在溶液中表征游离病毒体上所有反应物（例如抗体、可溶性 CD4）的表位暴露。最近，我们扩展了这种方法，以便我们可以结合荧光共振能量转移（FRET）同时使用多种抗体探测病毒体，这种新颖的方法可以检测单个病毒体上的多个表位暴露。它同时揭示了两个表位探针之间的构象动力学，最重要的是，这些测量是使用我们的方法完成的，没有病毒体的遗传或外部扰动，因此，我们可以收集有关病毒体的结构和抗原动力学的前所未有的信息。 HIV 包膜，这将为抗病毒药物和抗 HIV 免疫的产生提供新的见解： 目标 1）表征单个病毒颗粒相关 HIV 三聚体上的多个表位的暴露。 2) 检查 HIV 病毒粒子上 gp120 的构象动力学 目标 3) 表征配体与可溶性裂解三聚体结合之前和过程中的构象灵活性。