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

项目总结 从感染细胞组装和释放HIV-1是病毒复制周期中的关键步骤。艾滋病病毒 组装由病毒编码的Gag多聚蛋白驱动。质膜弯曲成球状 芽、RNA基因组的包装(GRNA)和包膜糖蛋白(Env)的掺入都在其中 聚集和萌芽的关键事件。释放依赖于宿主编码的ESCRT蛋白，它 都是被塞进脖子里招募的。尽管在理解结构和细胞方面取得了长足的进步 关于艾滋病毒组装的机制，有几个基本问题仍然没有得到回答。目前还不清楚是如何做到的 GRNA的特异性包装已经实现，也不清楚Env是如何被整合到病毒膜上的。 ESCRT蛋白切断膜颈部并释放病毒粒子的分子机制 仍然不为人知。GAG由四个结构域组成：基质(MA)、衣壳(CA)、核衣壳(NC)和p6。体量 结合质膜脂质PI(4，5)P2，被认为在Env的掺入中起关键作用。CA表格 支撑薄膜弯曲并定位剩余区域的晶格。NC与RNA相对结合 本身不是特定的，但当适当地支架在膜上时，特定地包装二聚体 GRNA。最后，p6包含招募ESCRT切割机制的晚期结构域基序。我们的实验室已经采取了 认为与Gag结构域的多个低亲和力相互作用是由 可变形的膜，调节Env掺入、gRNA包装和ESCRT的高度特异性事件 补充和膜断裂。我们率先使用了重组全长肉豆蔻酰化艾滋病毒- 1个GAG与可变形的巨大单板层小泡(GUV)相结合。在以前的工作中，我们重新构建了 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 大量过剩的竞争对手RNA。我们最近采用了一种新技术来研究艾滋病毒的组装 并使用使用光学陷阱从GUVS中拉出的膜纳米管来释放。这个系统允许我们设置 用膜的曲率来定义一个值。通过将材料封装在GUV中，我们可以重建 ESCRT引起的膜断裂的拓扑结构。使用用于通过以下方式在GUV内对ATP进行光刻的系统 单泡紫外线照射，我们已经能够光学地触发膜断裂，可视化它，以及 测量与之相关的机械力。事实上，纳米管技术的一个主要优势是 与该过程中的每一步相关的机械力可以使用光学镊子读出。vbl.使用 这些工具，我们处于独特的位置来解决ESCRT介导的膜断裂的机制，以 了解GAG与ESCRT的耦合，定义gRNA和Env的包装方式，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.