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

在HIV-1感染的晚期，Gag多聚蛋白被转运到质膜（PM）， 组装件.在PM上的Gag靶向和组装取决于其基质（MA）之间的特异性相互作用。 结构域和磷脂酰肌醇4，5-二磷酸（PI（4，5）P2），一种位于内小叶的信号脂质， 首相在Gag组装的同时或之后，包膜（Env）蛋白被募集到PM中，用于 这是一个依赖于Gag组装的过程。若干条证据 提示Env的掺入是由Gag的MA结构域和细胞质的 gp 41的尾部（gp 41 CT），其机制仍有待阐明。十多年来，我们开创了 在分子水平上研究逆转录病毒（HIV-1，HIV-2，MLV和ASV）MA结构域如何影响HIV-1，HIV-2，MLV和ASV的方法。 Gag与脂质相互作用（例如，PI（4，5）P2）和膜，这是理解Gag组装的关键要求 并最终并入Env。表征gp 41 CT-MA相互作用的主要障碍是 重组gp 41 CT蛋白的不可用性和不能重建用于重组的适当条件。 互动发生。在这段资助期内，我们取得了突破性的进展，确定了三个- HIV-1 gp 41 CT的三维结构，并表征其与膜的相互作用。这一成就， 二十多年来，这项难以捉摸的任务填补了一个主要空白，提供了HIV-1最后一段的结构， 蛋白质组在这次更新中，我们的目标是阐明Gag介导的分子机制， 将HIV-1 Env招募到组装点。我们将检验MA结构域的三聚化 在gp 41 CT相互作用中起重要作用，因此Env募集到颗粒中。我们将雇用一名 一组结构生物学，生物物理学和生物化学工具，以产生一个大分子的图片， Gag的MA结构域与膜结合以及它如何与gp 41 CT相互作用。我们的具体目标是：（一）工程师 HIV-1 MA三聚体和六聚体，并表征其与膜的结合，（ii）表征相互作用 HIV-1 MA和gp 41 CT之间的关系，以及（iii）通过以下方法确定MA-gp 41（TM-CT）-膜复合物的结构： 单粒子冷冻电镜该提案基于以下坚实的前提：（1）25年的功能数据正在等待 为分子解释提供更清晰的见解Gag组装PM和Env的机制 公司，（2）与蛋白质，膜，结构和生物物理工作的高度技术技能集 工具，（3）强大的初步数据，以及（4）具有20-30年经验的优秀合作团队， cryo-EM、X射线晶体学和质谱法。因此，拟议的结构研究将提供 详细了解HIV-1 Gag组装对PM和Gag介导的Env掺入的影响。我们希望 该提议的结果将有助于开发抑制组装的新的抗病毒治疗剂， Env掺入并最终产生病毒。

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.