Structural basis for HIV-1 Gag interactions with cellular and viral constituents

在HIV-1感染的晚期，Gag多聚蛋白被运输到质膜(PM)，用于 集合。GAG在PM上的靶向和组装取决于其基质(MA)之间的特定相互作用 结构域和磷脂酰肌醇4，5-二磷酸(PI(4，5)P2)，一种定位于黄瓜内叶的信号脂质 首相同时或在GAG组装之后，包膜(Env)蛋白被招募到PM用于 整合到病毒颗粒中，这是一个依赖于Gag组装的过程。有几条证据 提示Env的掺入是通过GAG的MA结构域与细胞质之间的相互作用而介导的 Gp41(Gp41CT)的尾部，其作用机制尚待阐明。十多年来，我们一直在开拓 在分子水平上研究逆转录病毒(HIV-1、HIV-2、MLV和ASV)的MA结构域如何 GAG与脂类(如PI(4，5)P2)和膜相互作用，这是理解GAG组装的关键要求 最终成立Env公司。表征gp41CT-MA相互作用的一个主要障碍是 无法获得重组gp41CT蛋白，以及无法重建适当的条件 要发生的互动。在这段资金期间，我们取得了突破，确定了三项- HIV-1 gp41CT的空间结构及其与膜的相互作用。这一成就，以及 二十多年来难以捉摸的任务，通过提供HIV-1最后一段的结构填补了一个主要空白 蛋白质组。在这次更新中，我们着眼于阐明Gag介导的分子机制。 将艾滋病毒-1环境病毒招募到组装地点。我们将检验MA结构域的三聚化假设 在gp41CT相互作用中发挥重要作用，因此环境病毒重新募集到颗粒中。我们将聘请一名 由结构生物学、生物物理和生化工具组成的电池，以生成大分子图像 GAG的MA结构域与膜结合以及它如何与gp41CT相互作用。我们的具体目标是：(I)工程师 HIV-1 MA三聚体和六聚体及其与膜的结合；(Ii)相互作用的特征 在HIV-1MA和gp41CT之间，以及(Iii)通过以下方法确定MA-gp41(TM-CT)-膜复合物的结构 单粒子低温电磁。该建议基于一个坚实的前提：(1)25年的功能数据正在等待 为分子解释提供对PM上的GAG组装和环境机制的更敏锐的见解 结合，(2)处理蛋白质、膜、结构和生物物理的高技术技能 工具，(3)强大的初步数据，以及(4)具有20-30年以下经验的优秀协作团队 低温电子显微镜、X射线结晶学和质谱学。因此，拟议的结构研究将提供 详细了解HIV-1 Gag在PM上的组装和Gag介导的Env整合。我们希望 这项提案的结果将有助于开发抑制组装的新的抗病毒治疗剂， Env并入，并最终产生病毒。

During the late phase of HIV-1 infection, the Gag polyproteins are transported to the plasma membrane (PM) for assembly. Gag targeting and assembly on the PM is dependent on specific interactions between its matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a signaling lipid localized on the inner leaflet of the PM. Concurrent or subsequent to Gag assembly, the envelope (Env) protein is recruited to the PM for incorporation into virus particles, a process that is dependent on Gag assembly. Several lines of evidence suggest that incorporation of Env is mediated by interactions between the MA domain of Gag and the cytoplasmic tail of gp41 (gp41CT), a mechanism that remains to be elucidated. For over a decade, we have pioneered approaches to investigate at the molecular level how retroviral (HIV-1, HIV-2, MLV, and ASV) MA domains of Gag interact with lipids (e.g., PI(4,5)P2) and membranes, a key requirement for understanding Gag assembly and ultimately Env incorporation. A major barrier to characterizing gp41CT–MA interaction has been the unavailability of a recombinant gp41CT protein and the inability to reconstitute the proper conditions for the interaction to occur. During this funding period we have recorded a breakthrough by determining the three- dimensional structure of HIV-1 gp41CT and characterized its interaction with membrane. This achievement, an elusive task for over two decades, filled a major gap by providing the structure of the last segment of HIV-1 proteome. In this renewal, we set our sights on elucidating the molecular mechanism by which Gag mediates the recruitment of HIV-1 Env into assembly sites. We will test the hypothesis that trimerization of the MA domain plays an important role in gp41CT interaction and therefore Env recruitment into particles. We will employ a battery of structural biology, biophysical, and biochemical tools to generate a macromolecular picture of how the MA domain of Gag binds to membrane and how it interacts with gp41CT. Our specific aims are: (i) Engineer HIV-1 MA trimer and hexamer and characterize their binding to membranes, (ii) characterize the interactions between HIV-1 MA and gp41CT, and (iii) determine the structure of MA–gp41(TM-CT)–membrane complex by single-particle cryo-EM. This proposal rests on a solid premise of: (1) 25 years of functional data that is waiting for a molecular interpretation to provide sharper insights into Gag assembly on the PM and mechanisms of Env incorporation, (2) highly technical skill sets for working with proteins, membranes, structural and biophysical tools, (3) strong preliminary data, and (4) an outstanding collaborative team with 20-30 years of experience in cryo-EM, x-ray crystallography, and mass spectrometry. Therefore, the proposed structural studies will provide a detailed understanding of HIV-1 Gag assembly on the PM and Gag–mediated Env incorporation. We hope that the outcome of this proposal will help in the development of new antiviral therapeutic agents that inhibit assembly, Env incorporation and ultimately virus production.