Membrane Coupling and Dynamic Reorganization of Gag in Viral Budding

描述(申请人提供)：逆转录病毒多蛋白GAG是许多病毒病原体，如人类免疫缺陷病毒1型(HIV-1)和肿瘤病毒的基本因子，它促进后代病毒壳在与宿主质膜结合后萌发，导致形成未成熟的子粒子，进一步成熟为传染性病毒。膜发芽过程中涉及的大量分子相互作用导致了随后的分子重组的复杂性，这阻碍了我们对这些过程的理解。这种认识的缺乏反过来又阻碍了通过治疗干预措施来瞄准病毒颗粒繁殖的这一重要步骤。在这里，我们主要关注两个假设。(I)与脂质双层的GAG结合是分子相互作用的分级序列的结果 在蛋白质和质膜之间：膜结合区的静电引导到双层表面；MA与磷脂酰肌醇二磷酸PI(4，5)P2的脂特异性相互作用；以及在MA的N端插入疏水的肉豆蔻基锚定。(Ii)在HIV-1的全长Gag多聚蛋白中， 不同的GAG结构域之间的连接区是控制导致膜萌发的分子重组的关键。我们组建了一个跨学科的研究团队，他们结合了卡内基梅隆大学(Carnegie Mellon University)Lash博士团队中位于膜界面的蛋白质结构和动态特性的尖端生物物理表征能力，以及以国家癌症研究所(NCI)Rein博士为代表的Gag蛋白质表达、操作和表征方面的专业知识，该团队在表征蛋白质结构的亚细胞定位和组装产品的超结构形态方面也具有先进的能力。这些小组将密切合作，在目标1中确定在结合研究中控制MA与膜结合的因素，用中子散射进行结构表征，并用相关光谱定量测量蛋白质与脂质双分子层的结合动力学。通过这些工具，我们还旨在了解导致特定脂类募集到病毒外壳中的机制。在目标2下，我们描述了膜表面全长GAG的构象重组的起源和在实验和计算研究中的意义。我们的研究团队凭借其互补的专业知识，是唯一有资格提高我们对病毒外壳形成所涉及的分子过程的理解的团队。 这项工作的公共卫生意义在于对艾滋病毒-1病毒和其他逆转录病毒病原体复制步骤中的病毒包膜形成有一个坚实的了解。其更广泛的影响是提供了新的技术来表征与脂质双层相关的膜蛋白的结构，这些膜蛋白处于生理上相关的热无序状态。

DESCRIPTION (provided by applicant): The retroviral polyprotein Gag is the essential factor in a large number of viral pathogens, such as human immunodeficiency virus type 1 (HIV-1) and oncoviruses, that promotes the budding of progeny viral shells after binding to the host plasma membrane, leading to the formation of immature daughter particles which mature further into infectious viruses. A multitude of molecular interactions involved in membrane budding result in a complexity of the ensuing molecular reorganizations that impedes our understanding of these processes. This lack in understanding in turn prevents the targeting of this important step in the reproduction of virus particles with therapeutic interventions. Here, we focus on two hypotheses. (I) Gag binding to the lipid bilayer results from a hierarchical sequence of molecular interactions between the protein and the plasma membrane: Electrostatic steering of the membrane-binding domain, MA, to the bilayer surface; lipid-specific interaction of MA with the phosphatidylinositoldiphosphate PI(4,5)P2; and hydrophobic membrane insertion of a myristoyl anchor on MA's N-terminus. (II) In the full-length Gag polyprotein of HIV-1, the flexibility of the linker regions between distinct Gag domains is key for controlling the molecular reorganization that leads to membrane budding. We assembled a cross-disciplinary team of investigators who combine cutting-edge biophysical characterization capabilities of the structural and dynamic properties of proteins at membrane interfaces, located in Dr. Lashes' group at Carnegie Mellon University (CMU), with expertise in Gag protein expression, manipulation and characterization represented by Dr. Rein's group at the National Cancer Institute (NCI) which also has advanced capabilities in characterizing subcellular localization of protein constructs and the ultra- structural morphology of assembly products. These groups will work closely together to determine, in Aim 1, the factors that control the association of MA with membranes in binding studies, structural characterization with neutron scattering and quantitative measurements of the dynamics of protein association with lipid bilayers with correlation spectroscopy. With these tools, we also aim at understanding the mechanisms that lead to the recruitment of specific lipids into the viral shell. Under Aim 2, we characterize the origin and the implications of the conformational reorganization of full-length Gag at membrane surfaces in experimental and computational studies. Our team of investigators with its complementing expertise is uniquely qualified to boost our understanding of the molecular processes involved in viral shell formation. The public health relevance of this work lies in the development of a solid understanding of viral envelope formation in the replication step of HIV-1 virus and other retroviral pathogens. Its broader impact is to provide new techniques for the structural characterization of membrane proteins associated with lipid bilayers in their physiologically relevant, thermally disordered state.