Membrane Coupling and Dynamic Reorganization of Gag in Viral Budding

描述（由申请方提供）：逆转录病毒多聚蛋白Gag是大量病毒病原体（如人类免疫缺陷病毒1型（HIV-1）和肿瘤病毒）中的必需因子，其在与宿主质膜结合后促进子代病毒壳出芽，导致形成未成熟的子代颗粒，其进一步成熟为感染性病毒。膜出芽中涉及的大量分子相互作用导致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。这种缺乏了解反过来又阻碍了治疗干预针对病毒颗粒复制中的这一重要步骤。在这里，我们集中在两个假设。(I)Gag与脂质双层的结合是由分子相互作用的分级序列引起的 之间的蛋白质和质膜：静电转向的膜结合域，MA，双层表面;脂质特异性相互作用的MA与磷脂酰肌醇二磷酸PI（4，5）P2;和疏水膜插入的肉豆蔻酰锚对MA的N-末端。(II)在HIV-1的全长Gag多聚蛋白中， 不同Gag结构域之间的接头区域是控制导致膜出芽的分子重组的关键。我们组建了一个跨学科的研究人员团队，他们将位于卡内基梅隆大学（CMU）Lashes博士小组的膜界面蛋白质结构和动态特性的尖端生物物理表征能力与Gag蛋白表达的专业知识相结合，由国家癌症研究所（NCI）的Rein博士小组代表的操作和表征其还具有表征蛋白质构建体的亚细胞定位和组装产物的超微结构形态的先进能力。这些小组将密切合作，以确定，在目标1，控制MA与膜结合研究，结构表征与中子散射和定量测量的动态蛋白质与脂质双层相关光谱学的关联的因素。利用这些工具，我们还旨在了解导致特定脂质募集到病毒外壳中的机制。根据目标2，我们的特点的起源和影响的构象重组的全长Gag在膜表面的实验和计算研究。我们的研究人员团队凭借其互补的专业知识，具有独特的资格，以提高我们对病毒外壳形成所涉及的分子过程的理解。 这项工作的公共卫生相关性在于对HIV-1病毒和其他逆转录病毒病原体复制步骤中病毒包膜形成的深入了解。其更广泛的影响是提供了新的技术与脂双层在其生理相关的，热无序状态的膜蛋白的结构表征。 公共卫生相关性：艾滋病毒等病毒在感染细胞中的繁殖取决于病毒蛋白Gag与细胞质膜的结合，细胞质膜被消耗以在复杂的分子重组序列中形成新的病毒外壳。在这个研究项目中，我们将确定导致病毒外壳形成的整个过程，因为对这个过程的彻底了解对于开发新的治疗策略来干扰感染细胞中的病毒形成是有价值的。

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. PUBLIC HEALTH RELEVANCE: The multiplication of viruses such as HIV in infected cells depends on the association of a viral protein, Gag, with the cell's plasma membrane which is consumed to form new viral shells in a complex sequence of molecular reorganizations. In this research project, we will determine the overall process that leads to the formation of viral shells because a thorough understanding of this process will be valuable for the development of new therapeutic strategies for interfering with virus formation in infected cells.