Computational and Biophysical Analysis of the Filovirus Matrix Protein System

项目摘要 埃博拉(EBOV)和马尔堡(Marv)丝状病毒导致人类严重出血热 高达90%的死亡率。他们的基因组只有七个基因，包括病毒基质蛋白 VP40在哺乳动物细胞中表达时，足以产生病毒样颗粒(VLP) 由不同的蛋白质-蛋白质(PPI)和蛋白质-脂质(PLI)相互作用介导，满足不同的 在病毒生命周期中扮演重要角色，使VP40成为蛋白质的“瑞士军刀”。引人入胜的 VP40的动态平衡和VLP作为直接观测模式系统的可用性 在BSL4实验室之外使VP40成为严格研究其生物物理基础的独特系统 这些研究的意义进一步增强。 因为VP40是病毒通过人类时最保守的蛋白质，但利用VP40 作为潜在的药物靶点，如果不了解 Stahelin和Wiest实验室，建立在彼此建立的合作和 结构生物物理学方法来研究这项资助的中心假设：域间 VP40的相互作用是病毒生命周期中VP40结构的关键调节因子。在两个具体的 我们将(I)确定VP40二聚体、六聚体和八聚体的生物物理机制 在硅胶、体外和人类细胞中的形式，以及(Ii)确定VP40在人类中如何产生突变 在暴发过程中以及在病毒传播过程中对VP40有贡献的动物 构象变化和重排成其单独的寡聚体形式。 这些问题将使用紧密集成的方法进行研究，使用多尺度分子 侧向和氢-重离子交换，VLP的细胞成像以及更传统的 超速离心、固相微萃取等生物物理实验测定药物结合常数 来自EBOV和MARV的野生型VP40以及相关突变体。这是一种创新和融合 方法不仅将提供对结果的仔细验证，还将提供对 PPI和PLI控制着许多时间和长度范围内的齐聚平衡，因此 使人们能够严格理解生物医学上非常重要的生物物理原理 丝状病毒蛋白将对理解其他PPI和PLI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.