A New System to Modulate Phosphatidylserine to Investigate Filovirus Budding

 描述（由申请方提供）：丝状病毒包括埃博拉病毒（EBOV）和马尔堡病毒（MARV），临床致死率可高达90%。这些病毒被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。丝状病毒具有丝状脂质包膜，尽管在30多年前被发现，但对它们如何从宿主细胞质膜复制和传播知之甚少。丝状病毒含有负义RNA基因组，其编码七种蛋白质。病毒基质蛋白VP40是七个基因之一，调节宿主细胞膜的组装和出芽。VP40完全是病毒脂质包膜的基础，但迄今为止，人们对VP40如何被运输到质膜并与人类细胞膜相互作用知之甚少。最近已显示VP 40在整个病毒生命周期中形成不同的蛋白质结构，因此它被称为Transformer蛋白。本申请的目的是解开介导VP40转运和脂质依赖性组装的复杂机制。中心假设是，VP40二聚体是敏感的磷脂酰丝氨酸（PS）浓度梯度沿着分泌途径，调节VP40的结构出现。我们的初步数据表明，生物膜的PS含量调节VP40膜结合和六聚化。VP40二聚体能够结合膜的PS含量类似的分泌囊泡，而VP40寡聚化是敏感的较高的PS含量与质膜的内小叶。该R21应用描述了提供VP40二聚体如何移动和转化为六聚体的生物化学图谱的实验。该提案的具体目标1将剖析PS含量在调节膜传感和运输中的VP40结构中的作用。我们将使用几种体外和细胞生物化学和生物物理测定来确定这些结构如何与细胞膜相互作用。为了了解VP40和脂质之间复杂的脂质相互作用的性质，我们将进一步开发一种可控的酶促PS消耗系统来研究VP40组装的活细胞动力学。具体目标2将阐明PS的作用和其他重要的生物物理性质的质膜在早期，中期和后期阶段的VP40组装。初步研究表明，VP40寡聚体对质膜流动性敏感，将评估VP40组装和出芽的定量实时动力学以检验中心假设。总的来说，这些研究应该对埃博拉病毒颗粒如何从细胞质膜形成产生新的重要机制见解。

 DESCRIPTION (provided by applicant): Filoviruses include Ebola virus (EBOV) and Marburg virus (MARV), which can have up to 90% clinical fatality. These viruses are classified as category A pathogens by the NIH as they pose a serious public health and national security risk. Filoviruses have a filamentous lipid-envelope and despite being discovered more than 30 years ago, not much is known on how they replicate and spread from the host cell plasma membrane. Filoviruses contain a negative sense RNA genome, which encodes for seven proteins. The viral matrix protein VP40, which is one of the seven genes, regulates assembly and budding from the host cell membrane. VP40 completely underlies the viral lipid envelope, but to date, little is known about how VP40 is trafficked to the plasma membrane and interacts with human cell membranes. VP40 has recently been shown to form different protein structures throughout the viral life cycle and thus it is referred to as a transformer protein. The goal of this application s to unravel the complex mechanisms that mediate VP40 transport and lipid-dependent assembly. The central hypothesis is that VP40 dimers are sensitive to the phosphatidylserine (PS) concentration gradient along the secretory pathway, which regulates the VP40 structures that arise. Our preliminary data indicate that the PS content of biological membranes regulates VP40 membrane binding and hexamerization. VP40 dimers are able to bind membranes with PS content similar to that of the secretory vesicles, while VP40 oligomerization is sensitive to a higher PS content associated with the inner leaflet of the plasma membrane. This R21 application describes experiments to provide a biochemical map of how VP40 dimers move and transform into hexamers. Specific Aim 1 of the proposal will dissect the role of PS content in regulating VP40 structures in membrane sensing and transport. We will use several in vitro and cellular biochemical and biophysical assays to establish how these structures interact with cellular membranes. In order to understand the nature of complex lipid interactions between VP40 and lipids, we will further develop a controllable enzymatic PS depletion system to study live cell dynamics of VP40 assembly. Specific aim 2 will elucidate the role of PS and other important biophysical properties of the plasma membrane in early, mid-, and late stage VP40 assembly. Preliminary studies demonstrate VP40 oligomers are sensitive to plasma membrane fluidity and quantitative real-time dynamics of VP40 assembly and budding will be assessed to test the central hypothesis. Taken together, these studies should produce new and important mechanistic insight into how an Ebola virus particle forms from the plasma membrane of cells.