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.

脂质包裹的病毒从宿主细胞中复制和芽，在那里他们获得脂质涂层。丝状病毒是脂质发育的病毒，具有丝状脂质 - eNvelope，尽管在40多年前被发现，但在如何获得脂质外套方面并不了解。这些病毒从宿主细胞的质膜上芽，并引起病毒出血热，死亡率高达90％。 FILOVIRES包括埃博拉病毒（EBOV）和Marburg病毒（MARV），它们被NIH归类为病原体类别，因为它们构成了严重的公共卫生和国家安全风险。这些病毒具有编码七个基因的负性RNA基因组。病毒基质蛋白VP40从宿主细胞膜中调节，是病毒脂质包膜的基础。 VP40是一种外周蛋白，也是这些病毒所需的唯一蛋白质才能形成丝状病毒，如颗粒，几乎与正宗病毒座几乎无法区分。由于对VP40如何与生物膜相互作用知之甚少，因此许多有关Fileovirus组装和发芽的基本问题仍未得到解决。初步研究表明，在质膜上有足够的病毒颗粒释放和VP40颗粒位移需要宿主细胞磷脂酶D。磷脂酶D是一种使用磷脂酰胆碱作为底物的酶来产生阴离子脂质磷脂酸（PA）。初步研究还表明，人类细胞中的VP40蛋白表达足以增加PA的细胞水平。该提案的中心假设是宿主磷脂酶D（PLD）在丝状病毒的晚期出芽中起着至关重要的作用。该R21应用描述了实验，以提供PLD活性和PA生成在Filovirus萌芽中的作用的粘性细胞和生化模型。具体目标1将研究PLD活性和PA生成在EBOV和MARV的细胞组装和萌芽中的作用。我们将阐明抑制该甘油磷脂合成以抑制丝状病毒的潜力。使用BSL-4合作者进行的实验还将检验该假设，以针对正宗的EBOV和MARV。具体目标2将研究PA稳定VP40低聚物的机制和VP40 PA结合的分子来源。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生成在质膜上稳定VP40低聚物中的作用。 PA结合的分子起源将使用脂质 - 蛋白质相互作用和已知VP40结构的强基本原理进行研究。综上所述，这些研究应产生有关细胞质膜如何形成的丝状病毒颗粒如何形成细胞膜的新的重要机械洞察力。