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

• 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
• 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

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，在宿主细胞中获得脂质外壳。丝状病毒是具有丝状脂质包膜的脂质包膜病毒，尽管在40多年前被发现，但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出芽，引起病毒性出血热，致死率高达90%。丝状病毒包括埃博拉病毒（EBOV）和马尔堡病毒（MARV），它们被NIH归类为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。这些病毒具有编码七个基因的负义RNA基因组。调节从宿主细胞膜出芽的病毒基质蛋白VP 40是病毒脂质包膜的基础。VP 40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，丝状病毒样颗粒与真正的病毒粒子几乎无法区分。由于对VP 40如何与生物膜相互作用知之甚少，因此关于丝状病毒组装和出芽的许多基本问题仍然没有答案。初步研究表明，宿主细胞酶，磷脂酶D，需要足够的病毒颗粒释放和VP 40颗粒在质膜上的位移。磷脂酶D是使用磷脂酰胆碱作为底物以产生阴离子脂质磷脂酸（PA）的酶。初步研究还表明，VP 40蛋白在人细胞中的表达足以增加PA的细胞水平。该建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起着至关重要的作用。该R21申请描述了提供PLD活性和PA产生在丝状病毒出芽中的作用的内聚细胞和生物化学模型的实验。具体目标1将研究PLD活性和PA产生在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合成以抑制丝状病毒出芽的潜力。与BSL-4合作者的实验也将针对真实的EBOV和MARV来测试这一假设。具体目标2将研究PA稳定VP 40寡聚体的机制以及PA与VP 40结合的分子起源。在我们的活细胞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产生在使VP 40寡聚体稳定在质膜上用于断裂过程中的作用。PA结合的分子起源将使用脂质-蛋白质相互作用的强有力的理论基础和已知的VP 40结构进行研究。总的来说，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机制见解。