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gp 41在HIV病毒进入中的分子作用。我们的长期目标是推进大的膜蛋白复合物的结构研究和协调的构象变化，通过它们的功能。该提案的目的是更好地定义导致HIV gp 41介导的生产性病毒膜融合的机制。本文提出的研究的基本原理是，gp 41在膜融合中的分子作用可以利用标记和分析的最新进展来阐明。新的试剂已经开发出来，便于标记的信封，并将允许该领域向前迈进。为了在概念验证的情况下测试这些新试剂，我们提出了以下具体目标：具体目标1。使用单颗粒融合试验确定参与HIV进入活细胞的大分子复合物的动力学。随着融合的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与衣壳核心的分离来测量融合孔形成的动力学。具体目标2。在病毒/细胞环境中定量HIV gp 41的捕获中间体。我们将使用共价反应肽结合到gp 41中间体和废除功能。我们将定量与总蛋白质相比捕获的中间体的量。这项研究意义重大，因为它将极大地增加在细胞环境中复制周期中包膜作用的结构知识。标签的进步将产生信息 具有揭示包膜内新型药物靶点的潜力。

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.