AVIAN RETROVIRUS STRUCTURE AND ASSEMBLY

禽逆转录病毒结构和组装

• 批准号: 6890863
• 负责人: Volker M. Vogt
• 金额: $ 34.85万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6890863
• 关键词: RNA directed DNA polymerase; Rous sarcoma virus; cell membrane; endopeptidases; fluorescence resonance energy transfer; gag protein; host organism interaction; integrase; liposomes; protein protein interaction; ubiquitin; virus assembly; virus morphology; virus protein

DESCRIPTION (provided by applicant): An important step in the life cycle of viruses is assembly of the virion (virus particle). For most retroviruses assembly takes place at the plasma membrane, where viral proteins and the RNA genome come together to form a budding nascent virion, which eventually becomes enveloped by membrane, pinching off from the cell. The long term goals of the proposed research are to further elucidate the process of retrovirus assembly, using the avian Rous sarcoma virus as a model system. The experiments, which emphasize biochemical approaches, are grouped under four specific aims. 1. Protein-RNA and protein-protein interactions. The experiments are designed to: (a) elucidate the role of the Gag NC domain, and of foreign protein interaction modules that can replace it, in assembly; (b) develop strategies for incorporating functional protease, reverse transcriptase, and integrase proteins into particles assembled in vitro; (c) further define the packaging sequence psi and how the NC domain interacts with psi, by selection of infectious viruses from a pool of proviruses with locally randomized sequences; (d) explore Gag-Gag interactions in living cells using fluorescence resonance energy transfer with GagGFP. 2. Protein-membrane interactions in assembly. The proposed experiments will: (a) define the parameters and biological relevance for Gag and MA binding to liposomes, and explore methods for reconstituting a membrane around immature viral cores or particles assembled in vitro; (b) determine the origin of the lipid raft-like composition of viral membranes, and with collaborators define the properties of GagGFP budding sites on the membrane, using single particle tracking in real time. 3. Late functions in assembly. The proposed experiments will: (a) identify late domain binding proteins and enzymes involved in ubiquitin metabolism that are incorporated into virions; (b) explore permeabilized cells as a model for studying the late steps in assembly. 4. Three dimensional structures of Gag proteins. Our structural biology collaborators will seek high resolution structures for: (a) CA and extended immature versions of CA; (b) the C-terminal CA domain extended through NC; (c) the portion of Gag including the late domain PPPY; (d) the monomeric PR domain as part of Gag. For all of these studies the principles that are learned should be applicable to HIV and to other retroviruses.

描述（由申请人提供）：生命周期中的重要一步 病毒是病毒粒子（病毒颗粒）的装配。对于大多数逆转录病毒 组装发生在质膜上，在那里病毒蛋白和RNA 基因组聚集在一起形成一个萌芽的新生病毒体， 被膜包裹，从细胞中夹出。的长期目标 建议的研究是进一步阐明逆转录病毒装配的过程， 使用禽类劳斯肉瘤病毒作为模型系统。这些实验， 强调生物化学方法，分为四个具体目标。1. 蛋白质-RNA和蛋白质-蛋白质相互作用。实验旨在： (a)阐明Gag NC结构域和外源蛋白相互作用的作用 模块，可以取代它，在组装;（B）制定战略， 掺入功能性蛋白酶、逆转录酶和整合酶 蛋白质在体外组装成颗粒;（c）进一步定义包装 序列psi以及NC结构域如何与psi相互作用，通过选择 来自具有局部随机序列的前病毒库的感染性病毒; (d)使用荧光共振探索活细胞中的Gag-Gag相互作用 GagGFP的能量转移。2.组装中的蛋白质-膜相互作用。的 拟议的实验将：（a）确定参数和生物相关性 的Gag和MA结合脂质体，并探索重建的方法， 体外组装的未成熟病毒核心或颗粒周围的膜;（B） 确定病毒膜的脂筏样组成的来源，和 与合作者一起定义了GagGFP出芽位点的性质， 膜，使用单粒子跟踪在真实的时间。3.迟交的职能 组装件.拟议的实验将：（a）鉴定晚期结构域结合 参与泛素代谢的蛋白质和酶， （B）探索透性化细胞作为研究晚期病毒颗粒的模型。 组装步骤。4. Gag蛋白的三维结构。我们 结构生物学合作者将寻求高分辨率结构：（a） （B）C-末端CA结构域延伸 （c）包括晚期结构域PPPY的Gag的部分;（d）包括晚期结构域PPPY的Gag的部分;（e）包括晚期结构域PPPY的Gag的部分;（f）包括晚期结构域PPPY的Gag的部分;（f）包括晚期结构域PPPY的Gag的部分。 单体PR结构域作为Gag的一部分。对于所有这些研究， 应该适用于艾滋病毒和其他逆转录病毒。