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

项目摘要 HIV-1从感染细胞中的组装和释放是病毒复制周期中的重要步骤。艾滋病毒 组装由病毒编码的Gag多蛋白驱动。质膜弯曲成球形 芽，RNA基因组（gRNA）的包装和包膜糖蛋白（Env）的掺入是其中之一 组装和萌芽的关键事件。释放依赖于宿主编码的ESCRT蛋白， 都是被塞哥塞到脖子上的尽管在理解结构和细胞方面取得了相当大的进展， 尽管艾滋病病毒的组装机制尚未明确，但仍有几个基本问题没有得到解答。我们不知道 尽管实现了gRNA的特异性包装，但也不清楚如何指导Env掺入到病毒膜中。 ESCRT蛋白切割膜颈并释放病毒体的分子机制也 仍然未知。Gag由四个结构域组成：基质（MA）、衣壳（CA）、核衣壳（NC）和p6。马 与质膜脂质PI（4，5）P2结合，并被认为在Env掺入中具有关键作用。CA形式 支撑膜弯曲并定位其余域的晶格。NC与RNA的结合相对 非特异性地自身，但当适当地支架在膜上时，特异性地包装二聚体， gRNA。最后，p6包含招募ESCRT断裂机制的晚期结构域基序。我们的实验室 认为与Gag结构域的多个低亲和力相互作用，其由一个 可变形膜，介导Env掺入、gRNA包装和ESCRT的高度特异性事件 募集和膜断裂。我们率先使用重组全长肉豆蔻酰化HIV- 1 Gag与可变形的巨大单层囊泡（GUV）结合。在以前的工作中，我们重建了 整个ESCRT级联的GUV上的Gag组件的招聘，我们展示了如何在 GUV膜NC获得了特异性结合背景中gRNA包装信号的能力 竞争者RNA的大量过剩。我们最近采用了一种新技术来研究艾滋病毒的组装 并使用光阱从GUV拉出的膜纳米管释放。这个系统允许我们设置 膜的曲率定义一个值。通过将材料封装在GUV中，我们可以重建 ESCRTs的膜断裂拓扑结构。在GUV内使用用于光释放ATP的系统， 单囊泡紫外线照射，我们已经能够光学触发膜断裂，可视化它， 测量与之相关的机械力。事实上，纳米管技术的一个主要优点是， 可以使用光镊读出与过程中的每个步骤相关联的机械力。使用 这些工具，我们在解决ESCRT介导的膜断裂机制， 理解Gag与ESCRT的偶联，以定义gRNA和Env如何包装，并确定 如果gRNA、Env和ESCRT相互影响。

PROJECT SUMMARY The assembly and release of HIV-1 from infected cells are essential steps in the viral replication cycle. HIV assembly is driven by the virally encoded Gag polyprotein. Bending of the plasma membrane into spherical buds, packing of the RNA genome (gRNA), and incorporation of the envelope glycoprotein (Env) are among the key events of assembly and budding. Release depends on the the host-encoded ESCRT proteins, which are recruited by Gag to the neck. Despite considerable progress in understanding structural and cellular mechanisms of HIV assembly, several fundamental questions remain unanswered. It is not understood how specific packing of gRNA is achieved, nor is it clear how Env incorporation into the viral membrane is directed. The molecular mechanism whereby the ESCRT proteins cut the membrane neck and release the virion also remains unknown. Gag consists of four domains: matrix (MA), capsid (CA), nucleocapsid (NC), and p6. MA binds to the plasma membrane lipid PI(4,5)P2 and is thought to have a key role in Env incorporation. CA forms a lattice that scaffolds membrane bending and positions the remaining domains. NC binds to RNA relatively non-specifically on its own, but when appropriately scaffolded on the membrane, specifically packages dimeric gRNA. Finally, p6 contains late domain motifs that recruit the ESCRT scission machinery. Our lab has taken the view that multiple low affinity interactions with Gag domains, which are brokered by the context of a deformable membrane, broker the highly specific events of Env incorporation, gRNA packaging, and ESCRT recruitment and membrane scission. We have pioneered the use of recombinant full-length myristoylated HIV- 1 Gag in conjunction with deformable giant unilamellar vesicles (GUVs). In previous work, we reconstituted the recruitment of the entire ESCRT cascade by Gag assemblies on GUVs, and we showed how in the context of the GUV membrane, NC acquired the ability to specifically bind the gRNA packaging signal in the background of a large excess of competitor RNA. We have recently adopted a new technology for studying HIV assembly and release using membrane nanotubes pulled from GUVs using an optical trap. This system allows us to set the curvature of the membrane to defined a value. By encapsulating materials inside the GUV, we can recreate the topology of membrane scission by ESCRTs. Using a system for photo-uncaging ATP within the GUV by single vesicle UV illumination, we have been able to optically trigger membrane scission, visualize it, and measure mechanical forces associated with it. Indeed, a major advantage of the nanotube technique is that the mechanical forces associated with each step in the process can be read out using the optical tweezers. Using these tools, we are in a unique position to resolve the mechanism of ESCRT-mediated membrane scission, to understand the coupling of Gag to the ESCRTs, to define how gRNA and Env are packaged, and to determine if gRNA, Env, and ESCRT interations impact one another.