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Insight into the Ebola virus glycoprotein fusion mechanism gleaned from the 2013-2016 epidemic GP-A82V variant

从2013-2016年流行的GP-A82V变种中洞察埃博拉病毒糖蛋白融合机制

基本信息

批准号：
10334830
负责人：
JEREMY LUBAN
金额：
$ 2.79万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-01 至 2021-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10334830
关键词：
Animals Antibodies Antibody Response Biochemical Biological Assay Biophysics Body Fluids Cells Cleaved cell Complex Computer Models Cryoelectron Microscopy Crystallization Data Dendritic Cells Disease Outbreaks Ebola Hemorrhagic Fever Ebola virus Engineering Epidemic Epidemiologic Factors Equilibrium Fluorescence Resonance Energy Transfer Funding Genetic Genome Genomic Centers for Infectious Diseases Genotype Glean Glycoproteins Human Immunologics In Vitro Individual Infection Institutes Measurement Modeling Molecular Conformation Monoclonal Antibodies Mucous Membrane Mutagenesis Mutation NPC1 gene National Institute of Allergy and Infectious Disease Natural Killer Cells Pathogenesis Pathogenicity Persons Pharmaceutical Preparations Property Proteins Reporting Research Personnel Resistance Resources Structure System Testing Tissues Universities Variant Viral Viral Pathogenesis Virion Virus Virus Replication atomic interactions base experimental study glycoprotein structure humanized mouse insight molecular dynamics monomer mouse model mutant neutralizing antibody novel novel therapeutics reconstitution reverse genetics single molecule structural glycoprotein tissue tropism tool trait transmission process viral transmission

项目摘要

The 2013–2016 Ebola virus (EBOV) disease epidemic was orders of magnitude larger than any previous EBOV outbreak. Preliminary data indicate that GP-A82V, an EBOV glycoprotein mutant that came to dominate the outbreak, increases infectivity in human cells. To elucidate the mechanism by which GP-A82V increases infectivity, and to clarify its significance for Ebola virus replication and transmission, we have assembled a team that leverages NIAID resources at the IRF-Fort Detrick and the Genomic Center for Infectious Diseases at The Broad Institute. Aim 1 will be to investigate the mechanism by which GP-A82V increases virion fusogenicity. Computer modeling suggests that GP-A82V destabilizes glycoprotein conformation. Mutations engineered based on the models will be tested for effects on infectivity, the reorganization of critical interactions as determined by molecular dynamics simulations, conformational equilibrium as determined by smFRET, novel assays for GP fusion, Cryo-EM of GP trimers, and crystal complexes with the NPC1 C-loop. Aim 2 will be to assess the effect of GP-A82V in the context of the EBOV Makona variant on infectivity in human cells in vitro and in humanized mice. We will generate a reverse genetic system for the ancestral EBOV Makona lineage and test the effect of GP-A82V on this background. Replication of WT and GP-A82V will be compared in U20S cells, in human dendritic cells, and in a novel humanized mouse model where the effect of GP-A82V on virus sequence adaptation to specific tissue compartments will be assessed. From these experiments we expect to clarify the significance of GP-A82V for viral replication and transmission, taking into account the genetic background of the EBOV and the species-specific effects of GP-A82V. Aim 3 will be to examine the effect of GP-A82V on neutralizing antibodies. Preliminary data indicate that GP-A82V is relatively resistant to neutralization by particular antibodies. Using a panel of monoclonal antibodies targeting different parts of GP, we will determine whether neutralization resistance is a general property of GP-A82V, or if this trait is specific to antibodies targeting particular regions of GP. If differential neutralization is observed with particular antibodies, the effect of these on viral titer will be tested in the humanized mouse model. We will also determine whether GP-A82V alters neutralization sensitivity to convalescent sera from Guineans infected early or later in the outbreak, and from individuals treated at Emory University. From these studies we hope to determine whether the antibody response to EBOV was different depending on whether a person was infected with virus bearing GP-A82 or GP-A82V. If differences in neutralization titer correlate with virus genotype it would contribute to understanding the factors that determine survival in an infected individual or the efficiency of transmission to people who come into contact with infected body fluids. Finally, these studies will provide valuable experimental tools that will inform our studies on GP structure and function.

2013 年至 2016 年埃博拉病毒 (EBOV) 疫情的流行程度比以往任何一次 EBOV 疫情都要大几个数量级。初步数据表明，GP-A82V（一种埃博拉病毒糖蛋白突变体，在此次疫情中占据主导地位）增加了人类细胞的感染性。为了阐明 GP-A82V 增加感染性的机制，并阐明其对埃博拉病毒复制和传播的重要性，我们组建了一个团队，利用 IRF-德特里克堡和博德研究所传染病基因组中心的 NIAID 资源。目标 1 是研究 GP-A82V 增加病毒体融合性的机制。计算机模型表明 GP-A82V 会破坏糖蛋白构象的稳定性。将测试基于模型设计的突变对感染性的影响、通过分子动力学模拟确定的关键相互作用的重组、通过 smFRET 确定的构象平衡、GP 融合的新测定法、GP 三聚体的冷冻电镜以及与 NPC1 C 环的晶体复合物。目标 2 是评估 EBOV Makona 变种背景下 GP-A82V 对体外人类细胞和人源化小鼠感染性的影响。我们将为祖先 EBOV Makona 谱系生成一个反向遗传系统，并测试 GP-A82V 在此背景下的效果。将在 U20S 细胞、人类树突状细胞和新型人源化小鼠模型中比较 WT 和 GP-A82V 的复制，其中将评估 GP-A82V 对病毒序列适应特定组织区室的影响。考虑到 EBOV 的遗传背景和 GP-A82V 的物种特异性效应，我们希望通过这些实验阐明 GP-A82V 对于病毒复制和传播的重要性。目标 3 是检查 GP-A82V 对中和抗体的影响。初步数据表明 GP-A82V 对特定抗体的中和具有相对抵抗力。使用一组针对 GP 不同部分的单克隆抗体，我们将确定中和抗性是否是 GP-A82V 的一般特性，或者该特性是否是针对 GP 特定区域的抗体所特有的。如果用特定抗体观察到差异中和，则将在人源化小鼠模型中测试这些抗体对病毒滴度的影响。我们还将确定 GP-A82V 是否会改变对疫情早期或后期感染的几内亚人以及埃默里大学治疗者的恢复期血清的中和敏感性。从这些研究中，我们希望确定对 EBOV 的抗体反应是否不同，具体取决于一个人是否感染了带有 GP-A82 或 GP-A82V 的病毒。如果中和滴度的差异与病毒基因型相关，则将有助于了解决定感染者生存的因素或传播给接触感染体液的人的效率。最后，这些研究将提供有价值的实验工具，为我们对 GP 结构和功能的研究提供信息。