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

2013-2016年的埃博拉病毒(EBOV)疫情比以往任何一次埃博拉病毒疫情都要大几个数量级。初步数据表明，EBOV糖蛋白突变体GP-A82V在疫情中占据主导地位，它增加了人类细胞的传染性。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意义，我们组建了一个团队，利用IRF-Fort Detrick和布罗德研究所传染病基因组中心的NIAID资源。目的1研究GP-A82V提高病毒粒子融合活性的机制。计算机模拟表明，GP-A82V破坏了糖蛋白构象的稳定。基于这些模型设计的突变将被测试对感染性的影响，通过分子动力学模拟确定的关键相互作用的重组，由smFRET确定的构象平衡，用于GP融合的新方法，GP三聚体的冷冻EM，以及与NPC1 C环的晶体络合物。目的2是在EBOV Makona变异株的背景下评估GP-A82V对体外培养的人细胞和人源化小鼠的感染性的影响。我们将为祖传的EBOV Makona血统建立反向遗传系统，并在此背景下测试GP-A82V的效果。WT和GP-A82V的复制将在U20S细胞、人类树突状细胞和新的人源化小鼠模型中进行比较，在该模型中，将评估GP-A82V对病毒序列适应特定组织间隔的影响。通过这些实验，我们希望通过考虑EBOV的遗传背景和GP-A82V的物种特异性效应来阐明GP-A82V对病毒复制和传播的意义。目的3是检测GP-A82V对中和抗体的影响。初步数据表明，GP-A82V相对抵抗特定抗体的中和。使用一组针对GP不同部分的单抗，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作用，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是否改变了在疫情爆发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人对恢复期血清的中和敏感性。通过这些研究，我们希望确定对EBOV的抗体反应是否取决于一个人是否感染了携带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，这将有助于理解决定感染者存活率的因素，或者决定接触受感染体液的人的传播效率。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.