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

基本信息

批准号：
10317727
负责人：
Robert Virgil Stahelin
金额：
$ 72.76万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10317727
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 Light Link Lipid Binding Lipids Mammalian Cell Marburgvirus Mediating Membrane Methods Modeling Molecular Molecular Conformation Mutate Mutation N-terminal Peptides 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 monomer mortality multitask mutant new therapeutic target pathogen peptidomimetics protein protein interaction protein structure function simulation tool

项目摘要

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.

项目概要埃博拉 (EBOV) 和马尔堡 (MARV) 丝状病毒会导致人类出现严重出血热他们的基因组仅包含七个基因，包括病毒基质蛋白，死亡率高达 90%。 VP40 在哺乳动物细胞中表达时足以产生病毒样颗粒 (VLP) 与活病毒粒子基本上没有区别，其形式为二聚体、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，实现不同的和在病毒生命周期中发挥着重要作用，使 VP40 成为蛋白质中的“瑞士军刀”。 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 成为一个独特的系统，可以严格研究生物物理基础病毒出芽以及 PPI 和 PLI 的总体意义进一步增强。因为 VP40 是病毒在人类中传播时最保守的蛋白质，但利用 VP40 如果不了解其物理基础，潜在的药物靶点就不可能成功 VP40 的寡聚化和功能。 Stahelin 和 Wiest 实验室在相互建立的合作基础上其他几个提供特定专业知识的合作者将使用计算、实验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。目标，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计算机、体外和人类细胞中形成，并且 (ii) 确定 VP40 的突变如何在人类中出现在爆发过程中以及在动物中病毒传播过程中都会产生 VP40 构象变化和重排成单独的寡聚形式。这些问题将通过使用多尺度分子的紧密集成的方法来研究 µs 时间尺度上的动力学模拟和计算上的自由能扰动方法侧和氢-氘交换、VLP 的细胞成像以及更传统的生物物理实验，例如超速离心和 SPR，以确定结合常数来自 EBOV 和 MARV 的野生型 VP40 以及相关突变体，这是创新和整合的。方法不仅会仔细验证结果，而且会提供对结果的详细见解 PPI 和 PLI 控制着多个时间和长度范围内的寡聚平衡，因此使人们能够严格理解生物物理学原理，这对于生物医学非常重要丝状病毒蛋白将对理解其他 PPI 和 PLI 产生重大影响。