Molecular details of HIV fusion revealed using novel gp41 labeling

描述（由申请人提供）：我们对HIV知识的一个关键空白仍然是包膜跨膜亚基gp41在HIV病毒进入中的分子作用。我们的长期目标是推进大型膜蛋白复合物的结构研究以及它们发挥作用的一致构象变化。本提案的目的是更好地定义导致由HIV gp41介导的多产病毒膜融合的机制。本文提出的研究的基本原理是gp41在膜融合中的分子作用可以通过最新的标记和分析来阐明。新的试剂已经开发出来，便于标记包膜，并将使该领域向前发展。为了在概念验证的情况下测试这些新试剂，我们提出以下具体目标：使用单颗粒融合测定法确定参与HIV进入活细胞的大分子复合物的动力学。随着融合发生和融合后事件的进行，我们将通过监测包膜亚基分离和从衣壳核心分离来测量融合孔形成的动力学。具体目标2。在病毒/细胞背景下定量捕获HIV gp41中间体。我们将使用与gp41中间体结合的共价反应性肽并取消其功能。我们将量化捕获中间体的数量，与总蛋白质相比。这项研究具有重要意义，因为它将大大增加包膜在细胞复制周期中作用的结构知识。标签技术的进步将产生信息

DESCRIPTION (provided by applicant): A critical gap in our knowledge of HIV remains the molecular role of the envelope transmembrane subunit, gp41, in HIV viral entry. Our long-term goal is to advance the structural studies of large membrane protein complexes and the concerted conformational changes through which they function. The objective of this proposal is to better define the mechanism that leads to productive viral membrane fusion mediated by HIV gp41. The rationale that underlies the research proposed herein is that the molecular role of gp41 in membrane fusion can be elucidated using the latest advances in labeling and analysis. Novel reagents have been developed that facilitate labeling of envelope and will allow the field to move forward. In order to test these new reagents in proof of concept situations we propose the following specific aims: Specific Aim 1. Determine the dynamics of the macromolecular complexes involved in HIV entry in live cells using a single particle fusion assay. As fusion occurs and post-fusion events proceed, we will measure the dynamics of fusion pore formation by monitoring envelope subunit separation and separation from the capsid core. Specific Aim 2. Quantify the trapped intermediate of HIV gp41 in the virus/cell context. We will use covalently reactive peptides that bind to the gp41 intermediate and abrogate function. We will quantify the amount of trapped intermediate as compared to total protein. The proposed research is significant because it will dramatically increase the structural knowledge of the role of envelope in the replication cycle in the cellular context. Advancements in labeling will produce information with the potential to reveal novel drug targets within envelope.