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

项目摘要 由HIV-1包膜糖蛋白[Env;三聚体（gp 160）3裂解为（gp 120/gp 41）3]介导的膜融合， 是病毒进入宿主细胞并建立感染的第一个关键步骤。成熟的Env峰含有 非共价结合的受体结合亚基GP 120和融合亚基GP 41各三个拷贝。一 广泛的生物化学和结构研究已经揭示了病毒膜融合的一般情况。 gp 120与主要受体CD 4和辅助受体的顺序结合导致大的、不可逆的结构改变。 gp 41中的重排，其驱动膜融合过程。尽管我们在这方面取得了巨大的进步， 在过去的二十年里，人们对HIV-1 Env结构的了解，主要是基于对其可溶性 尽管我们已经发现了一些片段，但我们仍然缺乏膜环境中全长Env的原子图像。一系列 最近的研究，我们已经确定了跨膜结构域，膜近端外部 区域，以及通过NMR模拟脂质双层的双胞中的HIV-1 Env的胞质尾的一部分。 出乎意料的是，我们发现这些区域在脂质双层的存在下都形成了有序的三聚体簇 并且它们中的任何一种的破坏都会降低膜融合效率并改变细胞的抗原结构。 整个环境，这表明他们发挥关键的结构和功能的作用。这些新发现提供了 确定脂质中重组的全长HIV-1 Env结构的强有力的科学前提 通过冷冻电子显微镜（cryoEM）观察纳米盘。我们假设跨膜和膜- HIV-1 Env的近端区域全部采用对稳定性至关重要的确定的寡聚体结构， 膜全长蛋白的功能和抗原性。我们将利用最近的进展 在cryoEM和纳米盘技术中，并计划确定全长Env蛋白的结构， 在脂质双层中重构，单独或与基质蛋白复合。我们的目标是视觉化的小说 完整Env蛋白在膜环境中的结构特征，以充分了解其 结构-功能和促进用于疫苗开发的基于Env的免疫原设计。我们会把 以下具体目标：1）我们将在以下背景下确定全长HIV-1 Env的结构 2）我们将确定膜中完整Env抗原差异的结构基础 在具有不同抗体敏感性的HIV-1分离株中，3）我们将确定全长的结构， HIV-1 Env与基质蛋白复合。

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.