Building mechanistic insight into evolvability of viral cell-entry functions

项目总结/摘要： 流感、埃博拉、登革热和寨卡只是过去或正在发生的大流行病的几种病毒， 威胁复发。所有这些病毒都将它们的感染性基因组包装在细胞膜中 由病毒蛋白质装饰的包膜，介导感染性细胞的进入。由于机械的压力， 由于体积小，病毒常常联合收割机将不同的蛋白质结合在一起，相互依存 功能协调发展的然而，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 蛋白质联合收割机将不同的蛋白质结构域与膜融合和靶细胞附着功能结合。 膜融合通过将病毒包膜与细胞融合将病毒基因组递送到细胞内部 膜的附着结构域将病毒带到融合靶标。它们还能稳定核聚变 结构域在细胞外环境中，这阻碍了内体（细胞内的融合位点）内的融合， 它们作为融合抑制物。因此，生产性细胞进入需要蛋白质的稳定性和蛋白质的稳定性之间的微妙平衡。 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压力， 进入蛋白调节从动物到人受体的偏好，并根据需要改变融合蛋白的稳定性 在人与人之间传播一个受到约束的系统是如何实现极端适应性的 环境的变化和机械经济的影响在我过去的工作中，我取得了前所未有的成就， 流感病毒膜融合的分子水平分辨率结合1）单病毒粒子实时成像 真实病毒体与平面膜双层的膜融合，2）计算和理论 对各种融合中间体的时间延迟分布建模，和3）基于结构的诱变。这 这项工作首次揭示了膜融合的适应性是如何通过结合 多个融合蛋白对病毒的作用。单个流感病毒上的数百种融合蛋白 颗粒接触靶膜，但只有几个相邻的介导融合。我已确定 独立的渠道可用于流感微调融合，其中两个影响可用的池， 病毒体表面上的活性融合蛋白或所需数量的融合蛋白邻居。在这一提议中， 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 的流感，和2）定义的限制进化的流感病毒细胞进入功能的约束。我会 在我的综合方法的基础上， 完整HA在真实病毒体上的附着/融合抑制功能，以及在接近天然的条件下， 纯化的内体。通过定义进化规则、入口蛋白功能变化及其相互依赖性， 我们可能会提高我们对具有高流行潜力的病毒株的预测能力。基本原则 针对流感病毒推导出的基因可能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.