Conformational Landscape of the HIV-1 Envelope Glycoproteins

项目概要/摘要 人类免疫缺陷病毒（HIV-1）进入宿主细胞是由包膜糖蛋白介导的 (Env)三聚体。Env三聚体由三个gp120外部糖蛋白组成，所述糖蛋白与 三种gp41糖蛋白，锚定在病毒膜上，具有长（~145个残基） 胞质尾Env是病毒表面唯一的病毒特异性分子，是宿主的主要靶点 中和抗体Env构象的灵活性对于病毒进入是必不可少的，并且也有助于病毒进入细胞内。 HIV-1逃避宿主抗体反应的能力。在病毒进入过程中，gp120与受体的结合， CD4和CCR5/CXCR4，触发亚稳态Env的构象变化，最终导致gp41- 介导的病毒和靶细胞膜的融合。病毒体上的HIV-1 Env可能至少 三种构象："闭合的"预触发构象（状态1）、"开放的" CD4结合构象（状态2）、"开放的" CD4结合构象（状态3）和"开放的" CD4结合构象（状态4）。 3），和一个中间的“部分开放”构象（状态2）。在受体接合之前，State-1 Env 构象在能量上是有利的，使得原代HIV-1菌株相对抵抗 潜在的中和抗体。CD4结合驱动Env从状态1到状态2，然后进入状态3， 前发夹中间体。State-3 Env与CCR5或CXCR4共受体的结合促进了 的高度稳定的gp41六螺旋束，导致病毒和靶细胞膜的融合。 多个观察结果表明，State-1 Env构象是亚稳定的，并且容易被 改变Env序列或从膜环境中提取Env。事实上，稳定的可溶性 已经显示了gp140三聚体或洗涤剂增溶的Env三聚体，其详细结构是可用的 采取类似状态2的构造拟议的工作将解决我们目前不完整的理解 的State-1 Env构象。HIV-1尽管变异性很大，但它是如何维持一个State-1 Env 构象？我们先前已经表明，多个Env氨基酸残基的变化可以破坏 一级构象。在所提出的研究中，我们将鉴定天然多态性Env残基，当 改变，稳定功能状态-1构象。我们将评估状态1稳定和状态1 Env相互作用的不稳定变化决定了病毒表型。这些研究的一个重点将是 gp41近膜外区（MPER）的构象与蛋白质表达之间的关系 Env胞外域的其余部分的构象。 State-1 Env构象的亚稳定性阻碍其在免疫过程中呈递给宿主免疫系统。 自然感染HIV-1和接种疫苗后。拟议的研究将为生产纯化的 HIV-1 Env三聚体稳定在State-1构象，这是广泛中和的主要靶标。 抗体和有效的小分子进入抑制剂。了解State-1 Env构象是如何 监管应加快Env导向疗法和疫苗的设计。

PROJECT SUMMARY/ABSTRACT The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the envelope glycoprotein (Env) trimer. The Env trimer consists of three gp120 exterior glycoproteins non-covalently associated with three gp41 glycoproteins, which are anchored in the viral membrane and which possess long (~145-residue) cytoplasmic tails. As the only virus-specific molecule on the viral surface, Env is the major target for host neutralizing antibodies. Env conformational flexibility is essential for virus entry and also contributes to the ability of HIV-1 to evade the host antibody response. During virus entry, the binding of gp120 to the receptors, CD4 and CCR5/CXCR4, triggers conformational changes in the metastable Env that ultimately result in gp41- mediated fusion of the viral and target cell membranes. The HIV-1 Env on virions potentially samples at least three conformations: a “closed” pretriggered conformation (State 1), an “open” CD4-bound conformation (State 3), and an intermediate “partially open” conformation (State 2). Prior to receptor engagement, the State-1 Env conformation is energetically favored, rendering primary HIV-1 strains relatively resistant to the binding of potentially neutralizing antibodies. CD4 binding drives Env from State 1 to State 2 and then into State 3, the prehairpin intermediate. Binding of the State-3 Env to the CCR5 or CXCR4 coreceptor promotes the formation of the highly stable gp41 six-helix bundle, resulting in the fusion of the viral and target cell membranes. Multiple observations indicate that the State-1 Env conformation is metastable and easily disrupted by alteration of Env sequences or extraction of Env from a membrane environment. Indeed, stabilized soluble gp140 trimers or detergent-solubilized Env trimers for which detailed structures are available have been shown to adopt a State-2-like conformation! The proposed work will address our currently incomplete understanding of the State-1 Env conformation. How does HIV-1, despite its tremendous variability, maintain a State-1 Env conformation? We have previously shown that changes in multiple Env amino acid residues can disrupt the State-1 conformation. In the proposed studies, we will identify naturally polymorphic Env residues that, when changed, stabilize the functional State-1 conformation. We will evaluate how State-1-stabilizing and State-1- destabilizing changes in Env interact to determine viral phenotypes. One focus of these studies will be the relationship between the conformation of the gp41 membrane-proximal external region (MPER) and the conformation of the rest of the Env ectodomain. The metastability of the State-1 Env conformation impedes its presentation to the host immune system during natural HIV-1 infection and following vaccination. The proposed studies will inform efforts to produce purified HIV-1 Env trimers stabilized in a State-1 conformation, which serves as the major target of broadly neutralizing antibodies and potent small-molecule entry inhibitors. Understanding how the State-1 Env conformation is regulated should expedite the design of Env-directed therapies and vaccines.