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

描述（由申请人提供）：HIV-1出芽和释放的生化、生物物理和结构机制项目摘要HIV从受感染的T细胞和巨噬细胞中出芽和释放是HIV复制周期中的重要步骤。HIV芽的形成需要病毒编码的Gag多聚蛋白，释放需要宿主编码的ESCRT蛋白。ESCRT切断狭窄的膜颈，其在细胞质分裂和管腔内膜出芽成内体中生理性地发生。我们的实验室已经纯化并荧光标记了全长HIV-1 Gag，所有的人ESCRT蛋白，以及相关的Mason-Pfizer猴病毒（M-PMV）的衣壳。我们在体外将人ESCRT蛋白有序组装到膜相关的HIV-1 Gag簇上。基于这套独特的试剂，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学细节。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，我们将继续通过VPS 4剖析它们的ATP依赖拆卸。2.使用结构易处理的模型病毒重建ESCRT介导的释放。与HIV-1一样，M-PMV通过ESCRT-I从质膜上出芽。M-PMV具有的优点是，原衣壳可以在溶液中预组装，允许我们将原衣壳组装与膜结合和出芽分离。我们已经开发了M-PMV作为体外重建ESCRT依赖性出芽的模型系统，并将使用该系统以前所未有的空间分辨率获得机制见解。3.阐明TSG 101、VPS 28和VPS 37以及UMA蛋白同种型如何组装成ESCRT-I复合物。在所有ESCRT组分中，ESCRT-I复合物在ESCRT机制向HIV-1出芽位点的募集中具有中心和重要作用。四种VPS 37亚型和多达五种在内体和病毒芽殖中具有不同作用的UMA蛋白的存在创造了一个复杂的画面。将确定代表性的人ESCRT-I核心复合物的结构，并将其用作理解ESCRT-I组装中的亚基选择性和HIV出芽中的功能的起点。

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.