Molecular details of HIV fusion revealed using novel gp41 labeling

使用新型 gp41 标记揭示 HIV 融合的分子细节

基本信息

批准号：
8707959
负责人：
Amy Lynn Jacobs
金额：
$ 18.79万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-01 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8707959
关键词：
AIDS/HIV problem Adsorption Affinity Binding Biological Assay Boxing Capsid Cell fusion Cell membrane Cells Complex Data Development Drug Design Drug Targeting Dyes Endocytosis Environment Event Fluorescence Fluorescence Microscopy Future Goals HIV HIV Envelope Protein gp120 HIV Envelope Protein gp41 Health Human Infection Knowledge Label Life Lipids Location Macromolecular Complexes Magnetism Measures Mediating Membrane Membrane Fusion Membrane Proteins Mission Modeling Molecular Monitor National Institute of Allergy and Infectious Disease Pathway interactions Peptides Pharmaceutical Preparations Preclinical Drug Evaluation Process Proteins Protocols documentation Reagent Research Role Signal Transduction Site Surface System Temperature Testing Time United States National Institutes of Health Vaccine Design Vaccines Viral Virus Work base disorder prevention human disease improved microbial novel particle prevent prophylactic protein complex public health relevance receptor research study small molecule time use vaccine development

项目摘要

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.

描述（由申请人提供）：我们对 HIV 知识的一个关键差距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膜蛋白复合物的结构研究以及它们发挥作用的协同构象变化。该提案的目的是更好地定义导致 HIV gp41 介导的高效病毒膜融合的机制。本文提出的研究的基本原理是，可以使用标记和分析的最新进展来阐明 gp41 在膜融合中的分子作用。新型试剂的开发有助于包膜标记，并将推动该领域向前发展。为了在概念验证情况下测试这些新试剂，我们提出以下具体目标：具体目标 1. 使用单粒子融合测定确定参与 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态。具体目标 2. 量化病毒/细胞环境中捕获的 HIV gp41 中间体。我们将使用与 gp41 中间体结合并消除功能的共价反应肽。我们将量化捕获的中间体与总蛋白质的数量。拟议的研究意义重大，因为它将极大地增加关于包膜在细胞复制周期中的作用的结构知识。标签技术的进步将产生信息具有揭示包膜内新药物靶点的潜力。