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 - 2016年埃博拉病毒（EBOV）疾病流行的数量级比以前的EBOV爆发大。初步数据表明，GP-A82V是一种主导爆发的EBOV糖蛋白突变体，增加了人类细胞的感染。为了阐明GP-A82V增加感染的机制，并阐明了其对埃博拉病毒复制和传播的重要性，我们组装了一个团队，该团队在IRF-Fort Detrick和基因组中利用NIAID资源的基因组中心在广阔研究所的感染性疾病中心。 AIM 1将是研究GP-A82V增加病毒体融合性的机制。计算机建模表明，GP-A82V破坏了糖蛋白构象的稳定性。将测试基于模型的突变对感染的影响，通过分子动力学模拟确定的临界相互作用的影响，由SMFret确定的构象等效，GP融合的新测定，GP三聚体的冷冻测定，以及与NPC1 C-Cloop的晶体复合物。 AIM 2将是在EBOV MAKONA变体中评估GP-A82V在体外和人源性小鼠中感染的影响。我们将为祖先Ebov Makona血统生成一个反向遗传系统，并在此背景下测试GP-A82V的效果。在U20S细胞，人树突状细胞和新型的人性化小鼠模型中，将比较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室的影响。从这些实验中，我们希望考虑到EBOV的遗传背景以及GP-A82V的规格特异性效应，从而阐明GP-A82V对病毒复制和传播的重要性。目标3将是检查GP-A82V对中和抗体的影响。初步数据，表明GP-A82V相对抗神经抗体具有抗性。使用针对GP不同部位的单克隆抗体，我们将确定神经硅耐药性是否是GP-A82V的一般特性，还是该性状是针对针对GP特定区域的抗体的特异性。如果使用特定的抗体观察到差异神经硅，则将在人源性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是否会在爆发中早期或晚些时候感染的几内亚人以及在埃默里大学接受治疗的个人中会改变神经敏感性。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，它将有助于理解确定受感染个体生存的因素，或向与受感染体液接触的人传播的效率。最后，这些研究将提供有价值的实验工具，这些工具将为我们关于GP结构和功能的研究提供信息。