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）疾病的流行比以往任何一次EBOV疫情都要大几个数量级。初步数据表明，GP-A82 V是一种EBOV糖蛋白突变体，它在爆发中占主导地位，增加了人类细胞的感染性。为了阐明GP-A82 V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-Fort Detrick和Broad研究所传染病基因组中心的NIAID资源。目的1将研究GP-A82 V增加病毒粒子融合性的机制。计算机模拟表明，GP-A82 V使糖蛋白构象不稳定。将测试基于模型工程化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、GP三聚体的Cryo-EM以及具有NPC 1 C环的晶体复合物。目的2将是评估GP-A82 V在EBOV Makona变体的背景下对体外人细胞和人源化小鼠中的感染性的影响。我们将为祖先EBOV Makona谱系产生反向遗传系统，并测试GP-A82 V在此背景下的作用。将在U20 S细胞、人树突状细胞和新型人源化小鼠模型中比较WT和GP-A82 V的复制，其中将评估GP-A82 V对病毒序列适应特定组织区室的影响。从这些实验中，我们期望阐明GP-A82 V对于病毒复制和传播的意义，考虑到EBOV的遗传背景和GP-A82 V的物种特异性作用。目的3将是检查GP-A82 V对中和抗体的作用。初步数据表明GP-A82 V对特定抗体的中和具有相对抗性。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否是GP-A82 V的一般特性，或者这种特性是否对针对GP特定区域的抗体具有特异性。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-A82 V是否改变了对恢复期血清的中和敏感性，这些血清来自疫情早期或晚期感染的几内亚人以及在埃默里大学接受治疗的个人。从这些研究中，我们希望确定对EBOV的抗体反应是否不同，这取决于一个人是否感染了携带GP-A82或GP-A82 V的病毒。如果中和滴度的差异与病毒基因型相关，则将有助于理解决定感染个体存活率的因素或传播给接触受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，将告知我们的GP结构和功能的研究。

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.