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：
10449192
负责人：
Bing Chen
金额：
$ 81.61万
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/10449192
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.

项目概要 HIV-1等包膜病毒进入宿主细胞的第一个关键步骤是病毒膜融合。病毒融合蛋白是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点。以前的研究揭示了许多病毒融合体的可溶性片段的融合前和融合后构象蛋白质，但对其融合肽、跨膜和近膜结构知之甚少脂质双层背景下的区域。有强有力的证据表明膜的功能作用融合中相互作用的区域，但关于它们如何发挥其功能的机制研究仍然很少。我们假设与 HIV-1 包膜相关的其他融合蛋白的膜相互作用区域蛋白质 (Env) 采用明确的寡聚结构，这对于稳定性、功能和稳定性至关重要。膜中全长蛋白质的抗原性。在我们之前完成的研究中资助期间，我们已经确定了 TM 的结构、膜近端外部区域和使用最新 NMR 技术模拟脂质双层的 bicelles 中 HIV-1 Env 的细胞质尾部。我们发现这些区域都形成有序的三聚体簇并且构象耦合，并且破坏它们可以减少融合并改变整个 Env 的抗原结构。在本申请中，我们建议申请我们的 NMR/bicelle 技术用于研究 SIV Env 的膜区域和最近出现的 SARS- CoV-2 刺突 (S)，并使用冷冻电子显微镜确定全长蛋白质的结构在脂质纳米盘中重构。我们将定义这些关键结构元件在膜融合中的作用通过深度诱变和功能测定来分析区域。我们将追求以下具体目标：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融合后安排的组成部分； 3）我们将确定全长SIV Env的结构和膜背景下的 SARS-CoV-2 S； 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。