Conformational Dynamics of HIV Envelope by Single Molecule Spectroscopy

 描述(由申请人提供)：人们普遍认为，艾滋病毒感染的建立和过程是由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的体液反应之间的相互作用决定的。因此，为了解艾滋病毒三聚体的结构、功能和抗原特性进行了密集的努力。这些信息有望为开发抗病毒对策提供洞察力，包括包膜靶向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是在游离病毒粒子的背景下进行的。然而，几条证据表明，病毒粒子上的HIV包膜三聚体在溶液中通过一系列构象转换，和/或在病毒附着过程中与多达三个CD4受体分子结合。这反过来又提出了关于病毒三聚体性质的某些可测试的假设。将在该项目中进行测试的一项研究认为，随着三聚体逐渐与CD4饱和，三聚体的灵活性和抗原性会逐渐改变。这个项目还探索了一个相关的假设，即某些构象允许中和抗体和/或中和抗体和非中和抗体的组合与单个三聚体结合。我们的初步研究表明，后一种情况经常发生在病毒粒子上，而用纯化的可溶性三聚体进行免疫化学研究的病毒捕获试验的数据表明，在没有非中和表位的情况下，中和表位表达在“天然”的三聚体上。对这些假说的准确评估需要一种方法来询问病毒粒子，因为它们自然存在于溶液中，而不需要进行广泛的技术操作。此前，我们报道了一种基于荧光相关光谱(FCS)的单分子方法，用于表征所有反应物(如抗体、可溶性CD4)在溶液中连续暴露于游离病毒粒子上的表位。最近，我们扩展了这一方法，以便我们可以结合荧光共振能量转移(FRET)用多个抗体同时探测病毒粒子。这一新的方法检测了单个病毒粒子三聚体上的多个表位暴露。它同时揭示了两个表位探针之间作为FRET函数的构象动力学。最重要的是，这种测量是使用我们的方法完成的，没有病毒粒子的遗传或外部扰动。因此，我们可以收集有关HIV包膜结构和抗原动力学的前所未有的信息，这将为抗病毒药物的产生和抗HIV免疫提供新的见解。我们提出了三个目标：目的1)表征多个表位在单个病毒粒子相关的HIV三聚体上的暴露。目的2)检测gp120在HIV病毒粒子上的构象动力学。目的3)研究配体与可溶性裂解三聚体结合前和结合过程中的构象柔性。

 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.