Biochemical, Biophysical, and Structural Mechanisms of HIV-1 Budding and Release

HIV-1出芽和释放的生物化学、生物物理和结构机制项目概述受感染的t细胞和巨噬细胞的出芽和释放是HIV复制周期的重要步骤。HIV芽的形成需要病毒编码的Gag多蛋白，而释放需要宿主编码的ESCRT蛋白。escrt切断狭窄的膜颈，这是在细胞质分裂和腔内膜出芽形成内体时发生的生理现象。我们的实验室纯化并荧光标记了全长HIV-1 Gag、所有人类ESCRT蛋白和相关Mason-Pfizer猴病毒（M-PMV）的衣壳。我们在体外将人类ESCRT蛋白有序组装到膜相关的HIV-1 Gag簇上。基于这组独特的试剂，本项目的具体目标如下：1。阐明HIV-1 Gag组装、脂质结构域形成、RNA结合和ESCRT募集之间的生物物理耦合。我们将剖析Gag组装、脂质结构域形成、RNA包装和ESCRT招募之间的相互作用。在分析了escrt的组装之后，我们将继续剖析VPS4对它们的atp依赖性拆卸。2. 利用结构可处理的模型病毒重建escrt介导的释放。像HIV-1一样，M-PMV通过ESCRT-I从质膜上芽出。M-PMV的优点是原衣壳可以在溶液中预组装，使我们能够将原衣壳组装与膜结合和出芽分离。我们已经开发了M-PMV作为escrt依赖性出芽体外重建的模型系统，并将使用该系统在前所未有的空间分辨率下获得机制洞察。3. 阐明TSG101、VPS28、VPS37和UMA蛋白亚型如何组装成ESCRT-I复合体。在所有ESCRT成分中，ESCRT- 1复合体在ESCRT机制向HIV-1出芽位点的招募中起着核心和重要的作用。四种VPS37亚型和多达五种在内体和病毒出芽中发挥不同作用的UMA蛋白的存在创造了一个复杂的画面。研究人员将确定具有代表性的人类ESCRT-I核心复合体的结构，并以此为起点，了解ESCRT-I组装中的亚基选择性及其在HIV出芽中的功能。

DESCRIPTION (provided by applicant): Biochemical, Biophysical, and Structural Mechanisms of HIV-1 Budding and Release Project Summary The budding and release of HIV from infected T-cells and macrophages is an essential step in the HIV replication cycle. HIV bud formation requires the virally encoded Gag polyprotein, and release requires the host-encoded ESCRT proteins. The ESCRTs sever narrow membrane necks, which occur physiologically in cytokinesis and in intralumenal membrane budding into endosomes. Our laboratory has purified and fluorescently labeled full-length HIV-1 Gag, all of the human ESCRT proteins, and capsids of the related Mason-Pfizer Monkey Virus (M-PMV). We reconstituted the ordered assembly of the human ESCRT proteins onto membrane-associated HIV-1 Gag clusters in vitro. Building on this unique set of reagents, the specific aims of this project are as follows: 1. To elucidate in biophysical detail the coupling between HIV-1 Gag assembly, lipid domain formation, RNA binding, and ESCRT recruitment. We will dissect the interplay between Gag assembly, lipid domain formation, RNA packaging, and ESCRT recruitment. Having analyzed the assembly of ESCRTs, we will go on to dissect their ATP-dependent disassembly by VPS4. 2. To reconstitute ESCRT-mediated release using a structurally tractable model virus. M-PMV buds from the plasma membrane via ESCRT-I, like HIV-1. M-PMV has the advantage that procapsids can be pre- assembled in solution, allowing us to decouple procapsid assembly from membrane binding and budding. We have developed M-PMV as a model system for in vitro reconstitution of ESCRT-dependent budding, and will use the system to obtain mechanistic insight at unprecedented spatial resolution. 3. To elucidate how TSG101, VPS28, and VPS37 and UMA protein isoforms assemble into the ESCRT-I complex. Of all the ESCRT components, the ESCRT-I complex has a central and essential role in the recruitment of the ESCRT machinery to HIV-1 budding sites. The presence of four VPS37 isoforms and up to five UMA proteins with different roles in endosomal and viral budding creates a complicated picture. The structure of a representative human ESCRT- I core complex will be determined and used as the starting point to understand subunit selectivity in ESCRT-I assembly and function in HIV budding.