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

2013-2016年埃博拉病毒（EBOV）疾病流行比以往任何埃博拉疫情都要大几个数量级。初步数据表明，在此次暴发中占主导地位的EBOV糖蛋白突变株GP-A82V增加了人细胞的传染性。为了阐明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，我们组建了一个团队，利用IRF-Fort Detrick和Broad研究所传染病基因组中心的NIAID资源。目的1将是研究GP-A82V增加病毒粒子融合原性的机制。计算机模拟表明GP-A82V破坏糖蛋白构象的稳定性。基于这些模型设计的突变将被测试对感染性的影响、由分子动力学模拟确定的关键相互作用的重组、由smFRET确定的构象平衡、GP融合的新检测、GP三聚体的冷冻电镜和具有NPC1 c环的晶体复合物。目的2将评估GP-A82V在EBOV Makona变异背景下对体外人和人源化小鼠细胞传染性的影响。我们将建立一个EBOV祖先马科纳谱系的反向遗传系统，并在此背景下测试GP-A82V的作用。将比较WT和GP-A82V在U20S细胞、人树突状细胞和一种新型人源化小鼠模型中的复制，在这种模型中，GP-A82V对病毒序列适应特定组织区室的影响将被评估。考虑到EBOV的遗传背景和GP-A82V的物种特异性作用，我们希望从这些实验中阐明GP-A82V对病毒复制和传播的意义。目的3将检验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的抗性。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GP- a82v的一般特性，还是这种特性是针对GP特定区域的抗体所特有的。如果观察到特定抗体的差异中和，这些抗体对病毒滴度的影响将在人源化小鼠模型中进行测试。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在埃默里大学接受治疗的个体的恢复期血清的中和敏感性。从这些研究中，我们希望确定抗体对EBOV的反应是否因感染GP-A82或GP-A82V病毒而不同。如果中和滴度的差异与病毒基因型相关，将有助于了解决定感染者存活的因素，或与受感染体液接触的人传播效率的因素。最后，这些研究将为我们对GP结构和功能的研究提供有价值的实验工具。

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.