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

描述（由申请方提供）：丝状病毒在灵长类动物中引起致死性出血热，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体以维持感染。产生新病毒体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很少。由于抑制宿主细胞质膜上新病毒体的形成是治愈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。为了形成感染性病毒体，新合成的病毒蛋白质和核衣壳形式的基因组RNA被转运到细胞膜以形成芽位点。芽位点的产生是治疗的主要目标，并且已经推测需要任何一系列的膜-蛋白质相互作用。我们展示了两种来自埃博拉病毒的称为基质蛋白的蛋白质; VP 40和VP 24与特定的脂质膜相关联，并诱导膜曲率的变化以产生病毒样颗粒（VLP）（新病毒体的模型）。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，通过研究病毒基质蛋白，可以获得许多关于外周蛋白组装机制的新信息，这些信息影响了多个科学团体。这项研究的主要目的是充分阐明丝状病毒科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VLP的机制。本研究的具体目的如下：1）确定基质蛋白VP 40的膜靶向和弯曲诱导机制; 2）确定细胞膜和F-肌动蛋白在靶向VP 24中的相互作用及其弯曲膜的能力; 3）确定VP 40和人Nedd 4 -1在产生VLP中的脂质依赖性相互作用性质。主要方法包括：（1）病毒蛋白相互作用的生物物理分析（2）利用荧光蛋白研究了VP 40和VP 24的细胞膜靶向性;（3）这些蛋白质在体外和哺乳动物细胞中的膜弯曲诱导特性的定性和定量：（4）用X射线和一系列荧光方法对膜弯曲过程的结构研究;（5）VP 40对人Nedd 4 -1蛋白质的劫持作用，（6）VP 24与F-actin的相互作用。总的来说，这些目标将提供一个全面的看法质膜为基础的病毒组装和新的病毒体的产生。公共卫生相关性：治疗或预防许多病毒感染的疗法缺乏，并且越来越担心国家或恐怖组织可能已经将病毒武器化以进行传播。拟议的研究将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体的第一个机制性见解，该病毒体是开发用于产生小分子抑制剂、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.