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

HIV-1 萌芽和释放的生化、生物物理和结构机制

基本信息

批准号：
10328869
负责人：
James H Hurley
金额：
$ 47.1万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-02-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10328869
关键词：
Address Adopted Affinity Antiviral Agents Binding Biochemical Biochemistry Biological Models Biology Biophysics Capsid Cell Size Cell membrane Cell physiology Cells Cellular biology Cholesterol Complement Complex Coupled Couples Coupling Cut protein Cytoplasmic Tail Cytosol Dimensions Drug resistance Encapsulated Event Evolution Genetic Materials Genome Geometry Glycoproteins Guide RNA HIV HIV Genome HIV-1 Human Kinetics Lead Length Life Cycle Stages Light Lighting Lipids Measures Mechanics Mediating Membrane Membrane Lipids Modeling Molecular Molecular Virology Nanotubes Neck Normal Cell Nucleocapsid Nucleotides Optics Outcome Pathway interactions Pharmaceutical Preparations Physiology Play Polymers Positioning Attribute Process Proteins RNA Radial Reaction Recombinants Regulation Role Signal Transduction Structure System Tail Techniques Testing Ursidae Family Vesicle Viral Viral Proteins Virion Virus Virus Replication Virus-like particle Work biochemical model biophysical model confocal imaging dimer gag Gene Products instrument laser tweezer lipid structure live cell imaging mechanical force membrane assembly membrane model new technology novel optical traps particle polymerization protein complex quantitative imaging reconstitution recruit scaffold success tool unilamellar vesicle

项目摘要

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.

项目概要 HIV-1 从受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) 的包装以及包膜糖蛋白 (Env) 的掺入等组装和萌芽的关键事件。释放取决于宿主编码的 ESCRT 蛋白，被Gag招募到脖子上。尽管在理解结构和细胞方面取得了相当大的进展尽管 HIV 组装机制尚未得到解答，但仍有几个基本问题尚未得到解答。不明白如何实现了 gRNA 的特异性包装，也不清楚 Env 是如何掺入病毒膜的。 ESCRT 蛋白切割膜颈并释放病毒颗粒的分子机制仍然未知。 Gag 由四个结构域组成：基质 (MA)、衣壳 (CA)、核衣壳 (NC) 和 p6。硕士与质膜脂质 PI(4,5)P2 结合，被认为在 Env 掺入中起关键作用。 CA 表格支撑膜弯曲并定位其余域的晶格。 NC 与 RNA 相对结合非特异性地单独存在，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。我们实验室采取了与 Gag 域的多个低亲和力交互的观点，这些交互是由 a 的上下文中介的可变形膜，促成 Env 掺入、gRNA 包装和 ESCRT 的高度特异性事件募集和膜分裂。我们率先使用重组全长肉豆蔻酰化 HIV- 1 Gag 与可变形巨型单层囊泡 (GUV) 结合。在之前的工作中，我们重构了通过 GUV 上的 Gag 组件招募整个 ESCRT 级联，我们展示了如何在 GUV膜，NC获得了在背景中特异性结合gRNA包装信号的能力大量过量的竞争者RNA。我们最近采用了一项新技术来研究艾滋病毒组装并使用光阱从 GUV 中拉出膜纳米管进行释放。该系统允许我们设置膜的曲率来定义一个值。通过将材料封装在 GUV 内，我们可以重新创建 ESRT 的膜分裂拓扑结构。使用 GUV 内光解笼 ATP 系统单囊泡紫外线照明，我们已经能够光学触发膜分裂，使其可视化，并且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每个步骤相关的机械力。使用有了这些工具，我们在解决 ESCRT 介导的膜分裂机制方面处于独特的地位，了解 Gag 与 ESCRT 的耦合，定义 gRNA 和 Env 的包装方式，并确定 gRNA、Env 和 ESCRT 相互作用是否相互影响。