Control of Retrovirus Assembly by Membrane Lipid Thermodynamic Activity.

描述（由申请人提供）：病毒侵入所有形式的生命，导致人类疾病，包括艾滋病毒-艾滋病。尽管病毒种类繁多，令人生畏，但所有包膜病毒，包括本文研究的HIV-1和劳斯肉瘤病毒（RSV），都必须与质膜的胞质小叶结合。体外研究的一个困难是缺乏质膜细胞质小叶的良好模型。另一个困难是真实的细胞在相对的外质小叶上可能具有数十纳米尺寸尺度的膜异质性。这两个实验问题都在这些拟议的研究中得到解决，与多组分模型细胞质混合物，可以耦合到相分离的脂质混合物在一个不对称的双层。本项目将探讨膜脂混合行为的三个方面如何与病毒Gag蛋白结合和组装相关：（1）膜结合磷脂酰丝氨酸的热力学活性如何受脂质组成的控制，以及这种PS活性如何与Gag结合相关？(2)Gag的另一个结合伴侣PI（4，5）P2的热力学活性是如何被其他膜脂质控制的，特别是，什么因素控制PI（4，5）P2结构域的形成？这些域是Gag组装的场所吗？(3)质膜是不对称的。相分离的小叶与胞质小叶的结合如何改变Gag的结合和组装？这项工作的一个主题是，膜脂结合或反应的趋势是由它们的热力学活性描述的，并且这种活性是由膜脂的所有组分控制的。 该混合物这种方法提供了预测能力，以描述病毒Gag结构蛋白与其脂质结合伙伴的关联：哪些膜因子控制膜结合病毒Gag蛋白之间的相互作用？总体策略是将脂质热力学活性的联合收割机测量与病毒蛋白结合和组装的测量相结合。荧光显微镜用于可视化PI（4，5）P2的结构域，并将这些结构域与测量的Gag结合和Gag-Gag组装到其病毒晶格中相关联。

DESCRIPTION (provided by applicant): Viruses invade all forms of life, causing diseases in humans including HIV-AIDS. Although the variety of viruses is daunting, all enveloped viruses including those studied here, HIV-1 and Rous Sarcoma Virus (RSV), must associate with the cytoplasmic leaflet of plasma membranes. One difficulty for in vitro studies is the lack of good models of the plasma membrane cytoplasmic leaflet. Another difficulty is that real cells can have membrane heterogeneities on the tens of nanometer size scale on the opposed, exoplasmic leaflet. Both of these experimental issues are addressed in these proposed studies, with a multicomponent model cytoplasmic mixture that can be coupled to a phase-separated lipid mixture in an asymmetric bilayer. This project will explore how three aspects of membrane lipid mixing behavior are related to viral Gag protein binding and assembly: (1) How is the thermodynamic activity of membrane-bound phosphatidylserine controlled by lipid composition, and how is this PS activity connected to Gag binding? (2) How is the thermodynamic activity of Gag's other binding partner, PI(4,5)P2 controlled by the other membrane lipids, and in particular, what factors control the formation of PI(4,5)P2 domains? Are these domains the sites of Gag assembly? (3) The plasma membrane is asymmetric. How does the presence of a phase-separated leaflet that is coupled to the cytoplasmic leaflet change Gag binding and assembly? A theme of this work is that the tendency of membrane lipids to bind or react is described by their thermodynamic activity, and this activity is controlled by all the components of the mixture. This approach provides predictive power to describe the associations of viral Gag structural proteins with their lipid binding partners: Which membrane factors exert control over the interactions among membrane-bound viral Gag proteins? The overall strategy is to combine measurements of lipid thermodynamic activity with measurement of virus protein binding and assembly. Fluorescence microscopy is used to visualize domains of PI(4,5)P2, and to correlate these domains with measured Gag binding and Gag-Gag assembly into its viral lattice.