Investigation of the role of phosphatidic acid metabolism in filovirus budding

磷脂酸代谢在丝状病毒出芽中的作用的研究

基本信息

批准号：
9979431
负责人：
Robert Virgil Stahelin
金额：
$ 20.57万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-01 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9979431
关键词：
Animal Model Antibodies Binding Biochemistry Biological Biological Assay Biological Models Biophysics Boston Category A pathogen Cell membrane Cell model Cells Cellular Assay Cellular Membrane Clinical Collaborations Complement Data Democratic Republic of the Congo Diacylglycerol Kinase Disease Outbreaks Drug Targeting Ebola virus Electron Microscopy Enzymes Escape Mutant FDA approved Family Fatality rate Filoviridae Infections Filovirus Frankfurt-Marburg Syndrome Virus Fright Generations Genes Genome Glycerophospholipids Human Human Cell Line In Vitro Investigation Kinetics Knock-out Label Lead Lecithin Life Cycle Stages Lipid Bilayers Lipid Binding Lipids Mammalian Cell Measurement Mediating Membrane Membrane Lipids Metabolism Microscopy Modeling Molecular Molecular Conformation Mutation N-terminal National Security Neck Peripheral Phosphatidic Acid Phospholipase D Play Preventive measure Process Proteins Public Health Publications RNA Recording of previous events Regulation Risk Role Small Interfering RNA Structure System Testing Therapeutic Time Transferase Uganda United States National Institutes of Health Universities Vaccine Clinical Trial Vaccines Viral Viral Hemorrhagic Fevers Viral Matrix Proteins Virion Virus Virus Replication Virus-like particle Western Africa biochemical model cohesion experience experimental study inhibitor/antagonist innovation insight mimetics pandemic disease particle protein expression protein protein interaction role model small molecule therapeutic development ubiquitin-protein ligase viral envelope lipids virus envelope

项目摘要

Lipid enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Filoviruses are lipid-enveloped viruses that have a filamentous lipid-envelope and despite being discovered more than 40 years ago, not much is known on how they acquire their lipid coat. These viruses bud from the plasma membrane of the host cell and cause viral hemorrhagic fever with up to a 90% fatality rate. Filoviruses include Ebola virus (EBOV) and Marburg virus (MARV), which are classified as category A pathogens by the NIH as they pose a serious public health and national security risk. These viruses harbor a negative sense RNA genome that encodes seven genes. The viral matrix protein VP40, which regulates budding from the host cell membrane, underlies the viral lipid envelope. VP40 is a peripheral protein and the only protein required from these viruses to form filamentous virus like particles that are nearly indistinguishable from authentic virions. Since little is known about how VP40 interacts with biological membranes, many fundamental questions about Filovirus assembly and budding remain unanswered. Preliminary studies demonstrate that a host cell enzyme, phospholipase D, is required for sufficient viral particle release and VP40 particle displacement at the plasma membrane. Phospholipase D is an enzyme that uses phosphatidylcholine as a substrate to generate the anionic lipid phosphatidic acid (PA). Preliminary studies also demonstrate that VP40 protein expression in human cells is sufficient to increase cellular levels of PA. The central hypothesis of this proposal is that the host enzyme phospholipase D (PLD) plays an essential role in the late stage budding of filoviruses. This R21 application describes experiments to provide a cohesive cellular and biochemical model of the role of PLD activity and PA generation in Filovirus budding. Specific Aim 1 will investigate the role of PLD activity and PA generation in the cellular assembly and budding of EBOV and MARV. We will elucidate the potential for inhibition of synthesis of this glycerophospholipid to inhibit Filovirus budding. Experiments with a BSL-4 collaborator will also test this hypothesis against authentic EBOV and MARV. Specific aim 2 will investigate the mechanism by which PA stabilizes VP40 oligomers and the molecular origin of PA binding by VP40. Quantitative measurements in our live cell BSL-2 surrogate system will determine the kinetics of VLP assembly and release and the role of PLD activity and PA generation in stabilizing VP40 oligomers at the plasma membrane for the scission process. The molecular origins of PA binding will be investigated using strong rationale of lipid-protein interactions and the known VP40 structures. Taken together, these studies should produce new and important mechanistic insight into how Filovirus particles form from the plasma membrane of cells.

脂质包膜病毒从宿主细胞复制并出芽，并在宿主细胞中获得脂质外壳。丝状病毒是一种脂质包膜病毒，具有丝状脂质包膜，尽管被发现已有 40 多年的历史，但人们对它们如何获得脂质外壳知之甚少。这些病毒从宿主细胞的质膜中出芽，引起病毒性出血热，死亡率高达 90%。丝状病毒包括埃博拉病毒（EBOV）和马尔堡病毒（MARV），它们被美国国立卫生研究院（NIH）列为A类病原体，因为它们构成严重的公共卫生和国家安全风险。这些病毒含有编码 7 个基因的负义 RNA 基因组。病毒基质蛋白 VP40 位于病毒脂质包膜下方，可调节宿主细胞膜的出芽。 VP40 是一种外周蛋白，也是这些病毒形成丝状病毒样颗粒所需的唯一蛋白，与真正的病毒体几乎没有区别。由于人们对 VP40 如何与生物膜相互作用知之甚少，因此有关丝状病毒组装和出芽的许多基本问题仍未得到解答。初步研究表明，宿主细胞酶磷脂酶 D 是病毒颗粒充分释放和 VP40 颗粒在质膜上位移所必需的。磷脂酶 D 是一种使用磷脂酰胆碱作为底物生成阴离子脂质磷脂酸 (PA) 的酶。初步研究还表明，人类细胞中 VP40 蛋白的表达足以增加细胞中 PA 的水平。该提议的中心假设是宿主酶磷脂酶 D (PLD) 在丝状病毒的后期出芽中发挥重要作用。该 R21 应用描述了一些实验，以提供 PLD 活性和 PA 生成在丝状病毒出芽中的作用的内聚细胞和生化模型。具体目标 1 将研究 PLD 活性和 PA 生成在 EBOV 和 MARV 的细胞组装和出芽中的作用。我们将阐明抑制这种甘油磷脂的合成以抑制丝状病毒出芽的潜力。 BSL-4 合作者的实验也将针对真实的 EBOV 和 MARV 检验这一假设。具体目标 2 将研究 PA 稳定 VP40 寡聚物的机制以及 VP40 与 PA 结合的分子起源。我们的活细胞 BSL-2 替代系统中的定量测量将确定 VLP 组装和释放的动力学，以及 PLD 活性和 PA 生成在稳定质膜上的 VP40 寡聚物以实现分裂过程中的作用。 PA 结合的分子起源将利用脂质-蛋白质相互作用的强有力的原理和已知的 VP40 结构进行研究。总而言之，这些研究应该对丝状病毒颗粒如何从细胞质膜形成产生新的、重要的机制见解。