Building mechanistic insight into evolvability of viral cell-entry functions

项目摘要/摘要： 流感、埃博拉、登革热和寨卡病毒只是导致过去或正在发生的大流行的几种病毒， 有复发的危险。所有这些病毒都将它们的感染性基因组包装在细胞衍生的膜中 装饰有病毒蛋白的信封，可以调节感染细胞的进入。由于机械方面的压力 经济由于其体积小，病毒往往结合在单一的不同的相互依赖的蛋白质中 功能。然而，它们保持着极强的适应性--它们在不断变化的条件下迅速进化。病毒进入细胞 蛋白质结合了不同的蛋白质结构域与膜融合和靶细胞附着功能。 膜融合通过将病毒包膜与细胞合并将病毒基因组传递到细胞内部 薄膜。附着结构域将病毒带到融合靶点。它们还用于稳定核聚变 细胞外环境中的区域，它阻碍了内小体内的融合(细胞内的融合部位)- 它们充当核聚变抑制因子。因此，生产细胞进入需要蛋白质的稳定性和微妙的平衡 功能可塑性。改变细胞进入的外部压力的一个来源是中和抗体 由免疫系统产生。另一个来源是人畜共患病适应的机械性压力，当 进入蛋白调节从动物到人类受体的偏好，并根据需要改变融合蛋白的稳定性 人与人之间的传播。一个受约束的系统如何实现极端的适应性 无论是环境的变化还是机械化的经济？在我过去的工作中，我取得了前所未有的成就， 结合1)单病毒粒子实时成像对流感薄膜融合的分子水平分辨率 真病毒粒子与平面膜双层的膜融合，2)计算和理论 对不同融合中间体的时延分布建模，以及3)基于结构的突变。这 这项工作首次揭示了膜融合的适应性是如何通过结合 病毒上多个融合蛋白之间的作用。一种流感病毒上的数百种融合蛋白 颗粒与靶膜接触，但只有几个相邻的颗粒参与融合。我已经确认了 可用于流感微调融合的独立途径，其中两个影响现有的 病毒粒子表面的活性融合蛋白或所需数量的融合蛋白邻居。在这份提案中， 我试图1)确定允许相互依赖的细胞进入功能适应的分子机制 以及2)定义限制流感病毒细胞进入功能进化的限制因素。这就做 以我的组合方法为基础，实现膜融合和膜的整体分子图像 完整的对真实的病毒粒子和在接近自然条件下的 纯化的内质体。通过定义进化规则、进入蛋白质功能变化以及它们之间的相互依赖， 我们可能会提高对具有高大流行潜力的病毒株的预测能力。基本原则 推论的流感病毒很可能会延伸到其他病毒细胞进入系统。

PROJECT SUMMARY/ABSTRACT: Influenza, ebola, dengue and zika are only a few of the viruses which caused past or ongoing pandemics and threaten recurrence. All of these viruses package their infectious genomes in a cell-derived membrane envelope decorated with viral proteins that mediate infectious cell entry. Due to pressures for mechanistic economy imposed by their small size, viruses often combine in a single protein different interdependent functions. Yet they retain extreme adaptability – they evolve rapidly under changing conditions. Viral cell-entry proteins combine distinct protein domains with membrane fusion and target cell attachment functions. Membrane fusion delivers viral genomes to the cell interior by merging the viral envelope with a cell membrane. The attachment domains bring viruses to the fusion target. They also serve to stabilize fusion domains in the extracellular environment, which retards fusion within endosomes (sites of fusion inside cells) – they act as fusion repressors. Thus productive cell entry requires a delicate balance of protein's stability and functional plasticity. One source of changing external pressures on cell entry are neutralizing antibodies produced by the immune system. Another source are mechanistic pressures of zoonotic adaptations, when entry proteins adjust preference from animal to human receptors, and alter fusion protein stability as required for human-to-human transmission. How is the extreme adaptability achieved by a system that is constrained both by the changing environment and mechanistic economy? In my past work, I achieved unprecedented, molecular-level resolution of influenza membrane fusion by combining 1) single-virion real-time imaging of membrane fusion of authentic virions with planar membrane bilayers, 2) computational and theoretical modeling of time-delay distributions to various fusion intermediates, and 3) structure-based mutagenesis. This work offered the first glimpse into how adaptability of membrane fusion might be achieved through combined action among multiple fusion proteins on a virus. Hundreds of fusion proteins on a single influenza virus particle contact the target membrane, but only several neighboring ones mediate fusion. I have identified independent avenues available to influenza to fine-tune fusion, two of which affect either the available pool of active fusion proteins on the virion surface or the required number of fusion-protein neighbors. In this proposal, I seek to 1) determine the molecular mechanisms allowing adaptation of the interdependent cell-entry functions of influenza, and 2) define the constraints limiting evolvability of the influenza virus cell-entry functions. I will build upon my combined approaches to enable a holistic molecular picture of membrane fusion and membrane attachment/fusion-repression functions of intact HAs on authentic virions and in near native conditions of purified endosomes. By defining evolutionary rules, entry-protein functional changes, and their co-dependence, we might improve our predictive ability for viral strains with high pandemic potential. Fundamental principles deduced for influenza are likely to extend to other viral cell-entry systems.