Investigation of the role of phosphatidic acid metabolism in filovirus budding

脂质包膜病毒在获得脂质外壳的宿主细胞中复制和发芽。丝状病毒是一种脂质包膜病毒，具有丝状的脂质包膜，尽管在40多年前被发现，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萌发，引起致死率高达90%的病毒性出血热。丝状病毒包括埃博拉病毒（EBOV）和马尔堡病毒（MARV），它们构成严重的公共卫生和国家安全风险，被国立卫生研究院列为A类病原体。这些病毒携带一个负义RNA基因组，编码7个基因。调控宿主细胞膜出芽的病毒基质蛋白VP40是病毒脂质包膜的基础。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质，这种颗粒与真正的病毒粒子几乎无法区分。由于对VP40如何与生物膜相互作用知之甚少，许多关于丝状病毒组装和出芽的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，需要足够的病毒颗粒释放和VP40颗粒在质膜上的位移。磷脂酶D是一种利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，VP40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重要作用。本R21应用程序描述了提供PLD活性和PA生成在线状病毒出芽中作用的内聚细胞和生化模型的实验。特异性目的1将研究PLD活性和PA生成在EBOV和MARV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验证这一假设。具体目标2将研究PA稳定VP40寡聚物的机制以及PA与VP40结合的分子起源。我们的活细胞BSL-2替代系统的定量测量将确定VLP组装和释放的动力学，以及PLD活性和PA生成在分裂过程中稳定质膜上VP40低聚物的作用。PA结合的分子起源将利用脂质-蛋白相互作用和已知VP40结构的强大原理进行研究。综上所述，这些研究应该为丝状病毒颗粒如何从细胞的质膜形成提供新的和重要的机制见解。

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.