Computational and Biophysical Analysis of the Filovirus Matrix Protein System

丝状病毒基质蛋白系统的计算和生物物理分析

• 批准号: 10669678
• 负责人: Robert Virgil Stahelin
• 金额: $ 70.81万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10669678
• 关键词: Address; Affect; Affinity; Animals; Behavior; Binding; Biological Models; Biophysical Process; Biophysics; Bioterrorism; C-terminal; Cell membrane; Cells; Collaborations; Communicable Diseases; Computer Analysis; Computer Models; Data; Deuterium; Development; Disease; Disease Outbreaks; Drug Targeting; Ebola; Ebola virus; Equilibrium; FDA approved; Filament; Filovirus; Free Energy; Genes; Genetic Transcription; Genome; Grain; Grant; Human; Hydrogen; In Vitro; Knowledge; Laboratories; Length; Life Cycle Stages; Link; Lipid Binding; Lipids; Mammalian Cell; Marburgvirus; Mediating; Membrane; Methods; Modeling; Molecular; Molecular Conformation; Mutate; Mutation; Mutation Detection; N-terminal; Process; Property; Protein Analysis; Protein Conformation; Proteins; Public Health; Regulation; Role; Side; Structural Models; Structure; Surface; System; Testing; Time; Ultracentrifugation; Validation; Viral; Viral Hemorrhagic Fevers; Viral Matrix Proteins; Viral Proteins; Virion; Virus; Virus Assembly; Virus-like particle; biophysical analysis; biophysical chemistry; biophysical properties; biophysical techniques; cellular imaging; dimer; drug development; druggable target; experimental study; fascinate; frontier; in silico; innovation; insight; molecular dynamics; molecular scale; monomer; mortality; multitask; mutant; new therapeutic target; pathogen; peptidomimetics; protein function; protein protein interaction; protein structure; simulation; stapled peptide; tool

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.

项目摘要 埃博拉（EBOV）和马堡（MARV）丝病毒引起人类严重的出血热 高达90％的死亡率。它们的基因组仅包含七个基因，包括病毒基质蛋白 VP40在哺乳动物细胞中表达时，足以产生类似病毒的颗粒（VLP） 与活病毒基本上没有区别。 VP40形成二聚体，六聚体和八聚体 由不同的蛋白质蛋白（PPI）和蛋白 - 脂质（PLI）相互作用介导的 在病毒生命周期中的重要作用，使VP40成为蛋白质的“瑞士军刀”。迷人的 VP40的动态平衡和VLP作为直接观察模型系统的可用性 在BSL4实验室之外，VP40成为严格研究生物物理基础的独特系统 病毒萌芽以及PPI和PLIS总体上。这些研究的意义进一步提高 因为VP40是通过人类通过病毒传递时最组成的蛋白质，但利用VP40 因为潜在的药物目标不太可能在不了解物理基础的情况下成功 VP40的低聚和功能。 Stahelin和Wiest Laboratories建立在既定的合作和 提供特定专业知识的其他几个合作者将使用计算，实验和 结构性生物物理学方法来研究该赠款的中心假设： VP40的相互作用是病毒生命周期中VP40结构的关键调节剂。在两个特定方面 目的，我们将（i）确定VP40二聚体，六聚体和八聚体的生物物理机制 在体外和人类细胞中形成的形式，（ii）确定人类中出现的VP40突变 在爆发过程中以及病毒通过期间的动物过程中有助于VP40 构象变化和重新排列为其单独的寡聚形式。 这些问题将使用多尺分子的紧密整合方法进行研究 µS时尺度和自由能扰动方法上的动力学模拟 侧面和氢 - 居民交换，VLP的蜂窝成像以及更传统的 生物物理实验，例如超速离心和SPR，以确定的结合常数 来自EBOV和MARV的WildType VP40以及相关的突变体。这个无效和整合 方法不仅会仔细验证结果，而且还提供了详细的见解 PPI和PLI在许多时间和长度范围内管理寡聚等效性脑 对生物医学上非常重要的生物物理原理有严格的了解 丝状病毒蛋白将对理解其他PPI和PLI产生重大影响。