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。尽管病毒的种类繁多，但所有包膜病毒，包括这里研究的HIV-1和劳斯肉瘤病毒（RSV），都必须与质膜的细胞质小叶相关。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，真正的细胞在相反的外质小叶上可能具有几十纳米尺度的膜异质性。这两个实验问题都在这些拟议的研究中得到解决，采用多组分模型细胞质混合物，可以耦合到不对称双分子层中相分离的脂质混合物。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控制，这种PS活性如何与Gag结合相关？(2) Gag的另一个结合伙伴PI(4,5)P2的热力学活性如何受到其他膜脂的控制，特别是什么因素控制PI(4,5)P2结构域的形成？这些结构域是Gag组装的位点吗？(3)质膜不对称。相分离小叶与细胞质小叶耦合的存在如何改变Gag结合和组装？这项工作的一个主题是膜脂结合或反应的倾向是由它们的热力学活性描述的，而这种活性是由所有的成分控制的

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.