Computational and Biophysical Analysis of the Filovirus Matrix Protein System

项目摘要 埃博拉（EBOV）和马尔堡（MARV）丝状病毒在人类中引起严重的出血热， 高达90%的死亡率它们的基因组只包含包括病毒基质蛋白在内的7个基因 当在哺乳动物细胞中表达时，足以产生病毒样颗粒（VLP）的VP 40 与活的病毒体基本上无法区分。VP 40形成二聚体、六聚体和八聚体 由不同的蛋白质-蛋白质（PPI）和蛋白质-脂质（PLI）相互作用介导， VP 40在病毒生命周期中起着重要作用，使VP 40成为蛋白质的“瑞士军刀”。迷人的 VP 40的动态平衡和VLP作为直接观测模型系统的可用性 使VP 40成为一个独特的系统，严格研究生物物理基础， 病毒出芽以及PPI和PLI。这些研究的意义进一步增加 因为VP 40是病毒通过人体时最保守的蛋白质，但是利用VP 40 作为一个潜在的药物靶点，如果不了解其物理基础， VP 40的寡聚化和功能。 Stahelin和Wiest实验室建立在彼此建立的合作基础上， 其他几个合作者提供具体的专业知识，将使用计算，实验和 结构生物物理学的方法来调查这个补助金的中心假设： VP 40的相互作用是病毒生命周期中VP 40结构的关键调节因子。在两个具体 目的，我们将（i）确定VP 40二聚体，六聚体和八聚体的生物物理机制 在计算机模拟、体外和人类细胞中形成，以及（ii）确定人类中出现的VP 40突变如何 在暴发过程中以及在病毒传播过程中的动物中， 构象变化和重排成其单独的低聚物形式。 这些问题将使用多尺度分子的紧密集成的方法进行研究 µs时间尺度上的动力学模拟和计算上的自由能微扰方法 侧和氢-氘交换，VLP的细胞成像以及更传统的 生物物理实验，如超离心和SPR，以确定 来自EBOV和MARV的野生型VP 40以及相关突变体。这种创新和整合 这种方法不仅可以对结果进行仔细的验证，而且还可以提供详细的见解， PPIs和PLIs在许多时间和长度尺度上控制低聚平衡，因此 使人们能够严格理解生物物理学原理， 丝状病毒蛋白，这将对理解其他PPI和PLIs产生重大影响。

Project Summary Ebola (EBOV) and Marburg (MARV) filoviruses cause severe hemorrhagic fever in humans with up to 90% mortality rates. Their genome contains only seven genes including the viral matrix protein VP40 which, when expressed in mammalian cells, is sufficient to produce virus-like particles (VLPs) that are essentially indistinguishable from live virions. VP40 forms dimers, hexamers and octamers mediated by different protein-protein (PPI) and protein-lipid (PLI) interactions that fulfill different and essential roles in the viral lifecycle, making VP40 a “swiss army knife” of proteins. The fascinating dynamic equilibria of VP40 and the availability of VLPs as a model system for direct observations outside of a BSL4 laboratory make VP40 a unique system to rigorously study the biophysical basis for viral budding as well as PPIs and PLIs in general. The significance of these studies is further increased because VP40 is the most conserved protein upon virus passage through humans, but exploiting VP40 as a potential drug target is unlikely to succeed without understanding the physical basis for oligomerization and function of VP40. The Stahelin and Wiest laboratories, building on established collaborations with each other and several other collaborators supplying specific expertise, will use computational, experimental and structural biophysics methods to investigate the central hypothesis of this grant: that interdomain interactions of VP40 are key regulators of VP40 structures during the viral life cycle. In two specific aims, we will (i) Determine the biophysical mechanisms by which VP40 dimer, hexamer and octamers form in silico, in vitro and in human cells and (ii) determine how mutations of VP40 that arise in humans during the course of an outbreak as well as in animals during passage of virus contribute to VP40 conformational change and rearrangement into its separate oligomeric forms. These questions will be studied using a tightly integrated approach using multiscale molecular dynamics simulations on the µs timescale and free energy perturbation methods on the computational side and hydrogen-deuterium exchange, cellular imaging of VLPs as well as more traditional biophysical experiments such as ultracentrifugation and SPR to determine the binding constants of wildtype VP40 from EBOV and MARV as well as pertinent mutants. This innovate and integrated approach will not only provide careful validation of the results, but also provide detailed insights into the PPIs and PLIs governing the oligomerization equilibria across many time- and lengths scale, thus enabling a rigorous understanding of the biophysical principles for a biomedically very important filovirus protein that will have a significant impact on understanding other PPIs and PLIs.