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

描述(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具有感染性的病毒粒子来维持感染。产生新的病毒粒子的过程涉及一系列的蛋白质相互作用，但关于这一过程的机制信息很少。由于抑制宿主细胞质膜形成新的病毒粒子是治疗和预防这些感染的途径，因此识别关键的相互作用和抑制模式是至关重要的。为了形成具有感染性的病毒粒子，新合成的病毒蛋白和核衣壳形式的基因组RNA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，它需要任何一系列膜-蛋白质相互作用。我们证明了来自埃博拉病毒的两种被称为基质蛋白的蛋白质：VP40和VP24与特定的脂膜结合，并诱导膜曲率的变化来产生病毒样颗粒(VLP)(一种新的病毒粒子模型)。这一发现和随后的研究将在病毒的生命周期中创造一种新的范式。此外，通过对病毒基质蛋白的研究，可以获得许多关于外周蛋白组装影响不止一个科学共同体的机制的新信息。这项研究的主要目的是全面阐明丝状病毒科基质蛋白与脂质相互作用的机制，以及它们从宿主细胞产生VLP的机制。这项研究的具体目标如下：1)确定基质蛋白VP40的膜靶向和曲率诱导机制；2)确定细胞膜和F-肌动蛋白在靶向VP24过程中的相互作用及其弯曲膜的能力；3)确定VP40和人Nedd4-1在VLPs生成过程中的脂依赖相互作用特性。采用的主要方法包括：(1)用单层、表面等离子共振、沉淀和停流分析方法分析病毒蛋白(VP40和VP24)与各种模型膜的相互作用；(2)利用荧光蛋白研究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定性和定量的膜弯曲诱导特性；(4)用X射线和一系列荧光方法研究膜弯曲过程的结构；(5)VP40在体外和哺乳动物细胞中劫持人蛋白Nedd4-1；(6)VP24与F-肌动蛋白的相互作用特性。总而言之，这些目标将提供一个基于质膜的病毒组装和新病毒粒子生成的综合视图。与公共卫生有关：治疗或预防许多病毒感染的治疗方法很少，人们越来越担心国家或恐怖组织可能已武器化病毒以供传播。这项拟议的研究将首次从机制上深入了解病毒蛋白如何与哺乳动物细胞膜相互作用产生新的感染性病毒粒子，这是开发小分子抑制剂、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.