Structure-function studies of the membrane-interacting domains of HIV-1 Env spike

HIV-1 Env 刺突膜相互作用域的结构功能研究

基本信息

批准号：
10326632
负责人：
JAMES Jeiwen CHOU
金额：
$ 83.42万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-20 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10326632
关键词：
2019-nCoV Acquired Immunodeficiency Syndrome Adopted Alanine Animal Model Architecture Biogenesis Biological Assay COVID-19 pandemic Cell fusion Cells Chimeric Proteins Computer Models Coupled Cryoelectron Microscopy Cytoplasmic Tail Data Analyses Detergents Disease Elements Environment Funding Goals HIV HIV-1 Infection Intervention Length Lipid Bilayers Lipids Macaca mulatta Membrane Membrane Fusion Membrane Proteins Molecular Conformation Mutagenesis Peptides Physiological Play Preparation Process Property Proteins Protocols documentation Research Resolution Role SARS-CoV-2 spike protein SIV Sampling Scanning Structure Technology Testing Therapeutic Transmembrane Domain Vaccines Viral Viral Fusion Proteins Virus Assembly Virus Diseases env Gene Products fascinate fight against insight nanodisc technology nanodisk novel protein folding protein structure reconstitution therapeutic development therapeutic target therapy development vaccine development vaccine trial virus envelope

项目摘要

Project Summary The first critical step for enveloped viruses, such as HIV-1, to enter host cells is viral membrane fusion. Viral fusion proteins are fascinating protein folding machineries capable of adopting completely different conformations during the fusion process; they are also important vaccine and therapeutic targets. Previous studies have revealed both pre- and post-fusion conformations of the soluble fragments of many viral fusion proteins, but less is known for structures of their fusion peptide, transmembrane and membrane-proximal regions in the context of lipid bilayers. There is strong evidence for functional roles of the membrane- interacting regions in fusion, and yet mechanistic studies on how they exert their functions remain scarce. We hypothesize that membrane-interacting regions of other fusion proteins related to HIV-1 envelope protein (Env) adopt defined oligomeric structures that are critical for the stability, function and antigenicity of the full-length proteins in membrane. In the studies that we completed during the previous funding period, we have determined the structures of the TM, membrane proximal external region, and cytoplasmic tail of HIV-1 Env in bicelles that mimic lipid bilayers using the latest NMR technology. We find that these regions all form well-ordered trimeric clusters and are conformationally coupled, and that disrupting them can reduce fusion and alter the antigenic structure of the entire Env. In this application, we propose to apply our NMR/bicelle technology to investigate the membrane regions of SIV Env and the recently emerged SARS- CoV-2 spike (S), and to use cryo-electron microscopy to determine structures of the full-length proteins reconstituted in lipid nanodiscs. We will define roles in membrane fusion of critical structural elements of these regions by deep mutagenesis and functional assays. We will purse the following specific aims: 1) we will investigate the membrane-interacting components of SIV Env; 2) we will investigate the membrane-interacting components in the postfusion arrangement; 3) we will determine structures of the full-length SIV Env and SARS-CoV-2 S in the context of membrane; 4) we will elucidate roles of the membrane-interacting domains of HIV/SIV Env and SARS-CoV-2 S in their stability, function and antigenicity.

项目摘要进入宿主细胞的包裹病毒（例如HIV-1）的第一步是病毒膜融合。病毒融合蛋白具有令人着迷的蛋白质折叠机器，能够采用完全不同的融合过程中的构象；它们也是重要的疫苗和治疗靶标。以前的研究揭示了许多病毒融合的可溶性片段的融合前和融合后构象蛋白质的融合肽，跨膜和膜 - 蛋白质的结构知之甚少脂质双层的区域。有充分的证据表明膜的功能作用融合中的相互作用区域，以及关于它们如何发挥其功能的机械研究仍然很少。我们假设与HIV-1信封有关的其他融合蛋白的膜相互作用区域蛋白质（Env）采用定义的寡聚结构，这对于稳定性，功能和功能至关重要膜中全长蛋白的抗原性。在我们以前完成的研究中资金期，我们已经确定了TM的结构，膜近端外部区域和 HIV-1 ENV中的细胞质尾部，使用最新的NMR技术模仿脂质双层。我们发现这些区域都形成了排序良好的三聚合物簇，并在构象上耦合，这破坏了它们可以减少融合并改变整个ENV的抗原结构。在此应用程序中，我们建议申请我们的NMR/BICELLE技术研究了SIV Env的膜区域以及最近出现的SARS- COV-2尖峰（S），并使用冷冻电子显微镜确定全长蛋白的结构在脂质纳米盘中重构。我们将在这些关键结构元素的膜融合中定义角色通过深诱变和功能测定的区域。我们将为以下具体目标提取：1）我们将研究SIV Env的膜相互作用成分； 2）我们将研究膜相互作用灌注后安排中的组成部分； 3）我们将确定全长SIV环境的结构和 SARS-COV-2在膜的背景下； 4）我们将阐明膜相互作用域的作用 HIV/SIV Env和SARS-COV-2 S的稳定性，功能和抗原性。