Lipid-Protein Interactions in Viral Assembly and Virus Like Particle Formation

描述（由申请人提供）：丝状病毒在灵长类动物中引起致命的出血热，目前尚无治愈或治疗方法。这些病毒的生命周期是一个复杂的过程，需要从宿主细胞膜产生感染性病毒粒子来维持感染。产生新病毒粒子的过程涉及一系列蛋白质相互作用，但关于这一过程的机制信息缺乏。由于抑制宿主细胞质膜形成新病毒粒子是治疗和预防这些感染的途径，因此确定关键的相互作用和抑制模式至关重要。为了形成传染性病毒粒子，新合成的病毒蛋白和基因组RNA以核衣壳的形式被运输到细胞膜上形成芽位。芽位的产生是治疗的主要目标，据推测需要任何一系列的膜蛋白相互作用。我们展示了两种来自埃博拉病毒的蛋白质，称为基质蛋白；VP40和VP24与特定的脂质膜结合，诱导膜曲率的变化产生病毒样颗粒（VLPs）（一种新的病毒粒子模型）。这一发现和随后的调查将在病毒的生命周期中创造一个新的范例。此外，通过对病毒基质蛋白的研究，可以获得影响多个科学界的外周蛋白组装机制的许多新信息。本研究的主要目的是充分阐明丝状病毒基质蛋白与脂质相互作用的机制基础及其从宿主细胞产生VLPs的机制。拟开展的具体研究目的如下：1)确定基质蛋白VP40的膜靶向和曲率诱导机制；2)细胞膜与F-actin靶向VP24的相互作用及其弯曲膜的能力的测定；3)测定VP40与人Nedd4-1在VLPs生成过程中的脂依赖性相互作用特性。使用的主要方法包括：(1)通过单层、表面等离子体共振、沉淀和停止流分析对病毒蛋白（VP40和VP24）与各种模型膜的相互作用进行生物物理分析；(2)利用荧光蛋白检测VP40和VP24的细胞膜靶向性；(3)这些蛋白在体外和哺乳动物细胞中诱导膜曲率的定性和定量特性；(4)利用x射线和一系列荧光方法研究膜曲率过程的结构；(5) VP40在体外和哺乳动物细胞脂质膜上劫持人类蛋白Nedd4-1；(6) VP24与F-actin的相互作用特性。总的来说，这些目标将为基于质膜的病毒组装和新病毒粒子的产生提供一个全面的观点。公共卫生相关性：治疗或预防许多病毒感染的疗法缺乏，人们越来越担心一些国家或恐怖主义集团可能将病毒武器化以进行传播。拟议的研究将首次提供关于病毒蛋白如何与哺乳动物细胞膜相互作用以产生新的感染性病毒粒子的机制见解，作为开发小分子抑制剂，疫苗或治疗性抗体的治疗方案的先决条件。

DESCRIPTION (provided by applicant): Filoviruses for which there is no cure or treatment cause fatal hemorrhagic fevers in primates. The life cycle of these viruses is a complex process requiring production of infectious virions from the host cell membrane to sustain the infection. The process of generating a new virion involves an array of protein interactions but there is a paucity of mechanistic information on this process. Because inhibition of new virion formation from the plasma membrane of the host cell is an avenue of curing and preventing these infections, identifying the key interactions and modes of inhibition are of utmost importance. For an infectious virion to form, newly synthesized viral proteins and genomic RNA in the form of nucleocapsids are transported to the cell membrane to form a bud site. Generation of the bud site is a prime target for therapy and has been speculated to require any array of membrane-protein interactions. We demonstrate two proteins from Ebola termed matrix proteins; VP40 and VP24 associate with specific lipid membranes and induce changes in membrane curvature to generate virus like particles (VLPs) (a model of new virions). This discovery and subsequent investigation will create a new paradigm in the life cycle of a virus. Moreover, from studying the viral matrix proteins much new information can be obtained on mechanisms of peripheral protein assembly impacting more than one scientific community. The primary objective of this proposed research is to fully elucidate the mechanistic basis of lipid-interactions by filoviridae matrix proteins and their mechanism of generating VLPs from host cells. The specific aims for the proposed research are as follows: 1) Determination of the membrane targeting and curvature inducing mechanism of the matrix protein VP40; 2) Determination of the interplay of cellular membranes and F-actin in targeting VP24 and its ability to curve membranes; 3) Determination of the lipid-dependent interaction properties of VP40 and human Nedd4-1 in the generation of VLPs. The principal methodologies to be used include: (1) the biophysical analysis of interactions of viral proteins (VP40 and VP24) with various model membranes by monolayer, surface plasmon resonance, sedimentation, and stop-flow analysis; (2) the cellular membrane targeting properties of VP40 and VP24 using fluorescent proteins; (3) the qualitative and quantitative membrane curvature inducing properties of these proteins in vitro and in mammalian cells; (4) the structural investigation of the membrane curvature process with X-ray and an array of fluorescence methodologies; (5) the hijacking of the human protein Nedd4-1 by VP40 on lipid membranes both in vitro and in mammalian cells; (6) the interaction properties of VP24 with F-actin. Collectively, these aims will provide a comprehensive view of the plasma membrane based viral assembly and generation of new virions. PUBLIC HEALTH RELEVANCE: There is a paucity of therapeutics for treatment or prevention of many viral infections and there is increasing concern countries or terrorist groups may have weaponized viruses for dissemination. The proposed studies will provide the first mechanistic insight into how viral proteins interact with mammalian cell membranes to generate new infectious virions serving as a prerequisite to the development of therapeutic protocols for generation of small-molecule inhibitors, vaccines, or therapeutic antibodies.