AVIAN RETROVIRUS STRUCTURE AND ASSEMBLY

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)的集合。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中的新生病毒粒子，最终成为 被膜包裹，从细胞中掐掉。的长期目标 拟议的研究是为了进一步阐明逆转录病毒组装的过程， 以禽劳斯肉瘤病毒为模型系统。这些实验，即 强调生化方法，归类于四个具体目标。1. 蛋白质-RNA和蛋白质-蛋白质相互作用。这些实验旨在： (A)阐明Gag NC结构域和外源蛋白质相互作用的作用 可以在组装中取代它的模块；(B)制定战略，以 整合功能性蛋白酶、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和NC结构域如何与psi相互作用，通过选择 来自具有局部随机化序列的前病毒池的传染性病毒； (D)利用荧光共振技术探索活细胞中Gag-Gag的相互作用 利用GagGFP进行能量转移。2.组装过程中蛋白质与膜的相互作用。这个 拟议的实验将：(A)确定参数和生物相关性 GAG和MA与脂质体结合，并探索重建 (B)体外组装的未成熟病毒核心或颗粒周围的膜； 确定病毒膜的脂筏状成分的来源，以及 与合作者一起定义GagGFP萌芽站点的属性 膜，使用单粒子实时跟踪。3.中的延迟函数 集合。拟议的实验将：(A)识别晚期结构域结合 参与泛素代谢的蛋白质和酶 病毒粒子；(B)探索通透性细胞作为研究晚期病毒的模型 装配中的步骤。4.GAG蛋白的三维结构。我们的 结构生物学合作者将寻找高分辨率的结构：(A) CA和扩展的未成熟版本的CA；(B)扩展的C端CA域 到NC；(C)GAG的包括后期域PPPY的部分；(D) 单体PR结构域作为GAG的一部分。对于所有这些研究，原则是 所学到的知识应适用于艾滋病毒和其他逆转录病毒。

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.