Avian Retrovirus Structure and Assembly

描述(申请人提供)：HIV-1是影响数千万人的持续大流行的罪魁祸首，其他逆转录病毒导致人类和动物疾病。更好地理解逆转录病毒复制的基本原理应该是开发更多的药物并最终进行免疫干预的基础。逆转录病毒生命周期中的一个关键步骤是从病毒蛋白、病毒RNA基因组和受感染宿主细胞的脂膜组装病毒颗粒。这项建议的重点是揭开逆转录病毒组装的几个方面，主要使用易于处理和研究充分的禽劳斯肉瘤病毒作为模型系统，但包括一些关于HIV-1蛋白的重点实验。主要的具体目标1是表征RSV和HIV-1内部结构蛋白GAG与膜的相互作用。这些实验将涉及：(1a)RSV和HIV-1中RAFT样病毒膜的起源，(1b)磷脂酰肌醇二磷酸(PIP2)在HIV-1Gag膜靶向中的作用，以及(1c)RSV Gag膜相互作用结构域(MA)的体内功能。主要的特定目标2是表征未成熟病毒颗粒组装中的蛋白质-蛋白质相互作用和蛋白质-RNA相互作用。这些实验将讨论：(2a)促进RSV Gag未成熟组装的短氨基酸序列的结构功能，(2b)RSV Gag，NC的RNA结合域促进蛋白质二聚从而导致组装的机制，(2c)六磷酸肌醇(IP6)在促进HIV-1 Gag在体外正确组装方面的作用，以及(2d)导致RSV基因组特定包装的Gag-RNA相互作用的性质。许多拟议的工作都是以生化方法为基础的，特别是依赖于我们实验室开发的体外组装系统。

DESCRIPTION (provided by applicant): HIV-1 is responsible for an ongoing pandemic that has affected tens of millions of people, and other retroviruses cause both human and animal diseases. A better understanding of the fundamental principles of retrovirus replication should serve as an underpinning for the development of additional Pharmaceuticals and eventually of immunological intervention. A critical step in the life cycle of a retrovirus is assembly of the virus particle from viral proteins, the viral RNA genome, and the lipid membrane from the infected host cell. The focus of this proposal is to unravel several aspects of retrovirus assembly, using mainly the tractable and well-studied avian Rous sarcoma virus as a model system, but including some focused experiments on HIV-1 proteins. Major Specific Aim 1 is to characterize the interaction of the RSV and HIV-1 internal structural protein, Gag, with membranes. The experiments will address: (1a) the origin of the raft-like viral membrane in RSV and HIV-1, (1b) the role of phosphatidyl inositol bisphosphate (PIP2) in membrane targeting of HIV-1 Gag, and (1c) the in vivo functions of the membrane-interacting domain (MA) of RSV Gag. Major Specific Aim 2 is to characterize the protein-protein interactions and protein-RNA interactions in assembly of an immature virus particle. The experiments will address: (2a) the structural function of a short sequence of amino acids that promotes immature assembly in RSV Gag, (2b) the mechanism by which the RNA binding domain of RSV Gag, NC, promotes protein dimerization that then leads to assembly, (2c) the role of inositol hexakisphosphate (IP6) in promoting proper assembly of HIV-1 Gag in vitro, and (2d) the nature of the Gag-RNA interactions leading to specific packaging of the RSV genome. Much of the proposed work is grounded in biochemical approaches, in particular relying on an in vitro assembly system developed in our lab.