Structure of HIV-1 envelope spike in the context of membrane

项目摘要 膜融合，由HIV-1包膜糖蛋白[env；三聚体(Gp160)3裂解为(gp120/gp41)3]， 是病毒进入宿主细胞并建立感染的第一个关键步骤。一个成熟的环境尖刺包含 非共价结合受体结合亚基gp120和融合亚基gp41各三个拷贝。一个 广泛的生化和结构研究揭示了病毒膜融合的概貌。 Gp120与主要受体cd4和辅助受体的顺序结合导致大的、不可逆的结构。 Gp41中的重排，这驱动了膜融合过程。尽管我们在这方面取得了巨大进步 近二十年来对HIV-1env结构的了解，主要是基于对其可溶性的研究 我们仍然缺乏膜环境中全长Env的原子图像。在一系列 最近的研究，我们已经确定了跨膜区的结构，膜近端的外部 区域，以及HIV-1Env胞质尾部的一部分，用核磁共振技术模拟脂质双层。 出乎意料的是，我们发现这些区域都在脂双层的存在下形成了有序的三聚体簇合物 而其中任何一种的破坏都会降低膜融合效率并改变病毒的抗原结构 整个环境，表明它们在结构和功能上发挥着关键作用。这些新的发现提供了一个 确定脂类重组HIV-1全长包膜结构的有力科学前提 纳米盘的冷冻电子显微镜(CryoEM)。我们假设跨膜和膜- HIV-1Env的近端区域都采用对稳定性至关重要的确定的寡聚结构， 膜全长蛋白的功能和抗原性。我们将充分利用最近的进展。 在低温电子显微镜和纳米盘技术中，并计划确定全长环境蛋白的结构， 在脂双层中重组，单独或与基质蛋白形成复合体。我们的目标是将小说形象化 膜环境下完整的Env蛋白的结构特征，以获得对其 结构-功能，并促进基于包膜病毒的免疫原设计，用于疫苗开发。我们会把钱花在 以下是具体目标：1)我们将在以下背景下确定全长HIV-1环境病毒的结构 膜，2)我们将确定膜上完整包膜抗原差异的结构基础 在抗体敏感性不同的HIV-1分离株中，以及3)我们将确定全长结构 HIV-1包膜蛋白与基质蛋白形成复合体。

Project Summary Membrane fusion, mediated by HIV-1 envelope glycoprotein [Env; trimeric (gp160)3 cleaved to (gp120/gp41)3], is the first critical step for the virus to enter host cells and establish infection. A mature Env spike contains three copies each of noncovalently-associated receptor-binding subunit gp120 and fusion subunit gp41. A general picture of viral membrane fusion has emerged from extensive biochemical and structural studies. Sequential binding of gp120 to the primary receptor CD4 and a coreceptor leads to large, irreversible structural rearrangements in gp41, which drive the membrane fusion process. Despite tremendous progress in our understanding of the structure of HIV-1 Env over the last two decades, largely based on studies of its soluble fragments, we still lack an atomic picture of the full-length Env in a membrane environment. In a series of recent studies, we have determined the structures of the transmembrane domain, membrane proximal external region, as well as a portion of the cytoplasmic tail of HIV-1 Env in bicelles that mimic lipid bilayers by NMR. Unexpectedly, we find that these regions all form well-ordered trimeric clusters in the presence of a lipid bilayer and that disruption of any of them reduces membrane fusion efficiency and alters the antigenic structure of the entire Env, suggesting that they play critical structural and functional roles. These new findings provide a strong scientific premise to determine the structure of the full-length HIV-1 Env reconstituted in lipid nanodiscs by cryo-electron microscopy (cryoEM). We hypothesize that the transmembrane and membrane- proximal regions of HIV-1 Env all adopt defined oligomeric structures that are critical for the stability, function and antigenicity of the full-length protein in membrane. We will capitalize on the recent advances in cryoEM and nanodisc technology and plan to determine structures of the full-length Env proteins, reconstituted in lipid bilayers, both alone or in complex with the matrix protein. Our goal is to visualize novel structural features of the intact Env proteins in the context of membrane, to gain a full understanding of their structure-function and to facilitate Env-based immunogen design for vaccine development. We will purse the following specific aims: 1) we will determine the structure of a full-length HIV-1 Env in the context of membrane, 2) we will determine the structural basis for antigenic differences of the intact Env in membrane among HIV-1 isolates with different antibody sensitivity, and 3) we will determine the structure of a full-length HIV-1 Env in complex with matrix protein.