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：
10541247
负责人：
JEREMY LUBAN
金额：
$ 81.93万
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：
2020
资助国家：
美国
起止时间：
2020-01-02 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10541247
关键词：
Animals Antibodies Antibody Response Biochemical Biological Assay Biophysics Body Fluids Cells Complex Computer Models Cryoelectron Microscopy 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 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 experimental study genetic selection glycoprotein structure humanized mouse insight molecular dynamics monomer mouse model mutant neutralizing antibody novel novel therapeutics reconstitution reverse genetics single molecule tissue tropism tool trait transmission process viral outbreak 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) 疾病的流行程度比以往任何一次 EBOV 爆发都要大几个数量级。 GP-A82V 增加传染性的机制，并阐明其对埃博拉病毒复制和传播的重要性，我们组建了一个利用 NIAID 资源的团队IRF-Fort Detrick 和博德研究所传染病基因组中心的目标 1 将研究 GP-A82V 增加病毒体融合性的机制，计算机模型表明 GP-A82V 会破坏基于突变的糖蛋白构象。将测试模型对传染性的影响、通过分子动力学模拟确定的关键相互作用的重组、通过确定的构象平衡smFRET、GP 融合的新型检测、GP 三聚体的冷冻电镜以及 NPC1 C 环晶体复合物将评估 EBOV Makona 变体背景下 GP-A82V 对人类细胞感染性的影响。我们将在体外和人源化小鼠中生成祖先 EBOV Makona 谱系的反向遗传系统，并测试 GP-A82V 在此背景下的复制效果。 WT 和 GP-A82V 将在 U20S 细胞、人类树突状细胞和新型人源化小鼠模型中进行比较，其中将评估 GP-A82V 对病毒序列适应特定组织区室的影响，我们希望从这些实验中阐明。考虑到 EBOV 的遗传背景和 GP-A82V 的物种特异性影响，GP-A82V 对病毒复制和传播的重要性将是检查 GP-A82V 的影响。 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，我们将确定中和抵抗是否是 GP-A82V 的一般特性。如果该特征针对针对 GP 特定区域的抗体，如果观察到特定抗体的差异中和，我们还将在人源化小鼠模型中测试这些对病毒滴度的影响。 GP-A82V 改变了对疫情早期或后期感染的几内亚人以及埃默里大学个体的恢复期血清的中和敏感性，我们希望通过这些研究确定对 EBOV 的抗体反应是否因人是否感染病毒而不同。如果中和效价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者身体的人的效率。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。