Conformational Dynamics of HIV Envelope by Single Molecule Spectroscopy

 描述（由申请方提供）：人们普遍认为，HIV感染的建立和病程是由病毒复制和对病毒包膜三聚体（介导宿主细胞附着和进入）的体液应答之间的相互作用决定的。因此，深入的努力已经指向理解HIV三聚体的结构、功能和抗原特性。这些信息有望揭示开发抗病毒对策的见解，包括基于抗包膜抗体的靶向抗病毒药物和疫苗。 HIV包膜三聚体的性质主要是在游离病毒体的背景下研究的。然而，一些证据表明，HIV包膜三聚体在病毒粒子上通过一系列构象在溶液中和/或当它们在病毒附着期间接合多达三个CD 4受体分子时转变。这反过来又暗示了关于病毒三聚体性质的某些可检验的假设。在本项目中将测试的一种观点认为，随着三聚体逐渐被CD 4饱和，三聚体的灵活性和抗原性逐渐变化。在该项目中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结合单个三聚体。我们的初步研究表明，后者的情况经常发生在病毒粒子，而从病毒捕获测定的免疫化学研究与纯化的可溶性三聚体的数据表明，中和表位表达的“天然”三聚体在非中和表位的情况下。这些假设的准确评估需要一种方法来询问病毒体，因为它们自然存在于溶液中，而无需广泛的技术操作。以前，我们报道了一种单分子方法的基础上使用荧光相关光谱（FCS），以表征表位暴露的游离病毒体与所有反应物（如抗体，可溶性CD 4）连续在溶液中。最近，我们已经扩展了这种方法，使得我们可以同时用多种抗体结合荧光共振能量转移（FRET）来探测病毒体。这种新方法检测单个病毒体三聚体上的多个表位暴露。它同时揭示了作为两个表位探针之间的FRET的函数的构象动力学。最重要的是，这些措施是使用我们的方法完成的，没有遗传或外部干扰的病毒粒子。因此，我们可以收集有关HIV包膜的结构和抗原动力学的前所未有的信息，这将为抗病毒药物和抗HIV免疫的产生提供新的见解。我们提出了三个目标：目标1）表征单个病毒体相关HIV三聚体上多个表位的暴露。目的2）研究gp 120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.