Enrichment of the State-1 Conformation of the HIV-1 Envelope Glycoprotein

HIV-1 包膜糖蛋白的 State-1 构象的富集

• 批准号: 10094191
• 负责人: JOSEPH G SODROSKI
• 金额: $ 75.08万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-17 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10094191
• 关键词: Antibodies; Antibody Response; Antigens; Binding; CCR5 gene; CXCR4 gene; Cell membrane; Cell surface; Cells; Cleaved cell; Complex; Cryoelectron Microscopy; Crystallization; Cytoplasm; Cytoplasmic Tail; Data; Development; Endoplasmic Reticulum; Evaluation; Glycoproteins; Goals; Golgi Apparatus; HIV; HIV Envelope Protein gp120; HIV-1; Immune system; Isoleucine; Link; Mass Spectrum Analysis; Mediating; Membrane; Molecular Conformation; Nucleocapsid; Peptides; Preparation; Process; Production; Proline; Resistance; Sampling; Shapes; Son of Sevenless Proteins; Structure; Surface; System; Testing; Vaccine Design; Vaccines; Viral; Virion; Virus; Work; cell envelope; crosslink; design; disulfide bond; flexibility; glycoprotein structure; glycosylation; gp160; immunogenicity; inhibitor/antagonist; neutralizing antibody; receptor; receptor binding; single-molecule FRET; small molecule; vaccine evaluation; virtual; virus envelope

PROJECT SUMMARY/ABSTRACT The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the envelope glycoprotein (Env) trimer ((gp120/gp41)3). As the only virus-specific molecule on the viral surface, Env is the major target for host neutralizing antibodies. During virus entry, binding to the receptors triggers conformational changes in the metastable Env that allow the fusion of viral and target cell membranes. Env conformational flexibility, which is important for virus entry, also contributes to HIV-1's ability to evade the host antibody response. Prior to receptor engagement, the HIV-1 Env trimer on virions can potentially sample three conformations: a metastable "closed" conformation (State 1), the "open" CD4-bound conformation (State 3), and an intermediate "partially open" conformation (State 2). Primary HIV-1 Envs are maintained in State 1 by multiple intramolecular and intersubunit interactions, rendering these Envs 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. The conformational flexibility of HIV-1 Env trimers has created challenges for structural studies and for the design of vaccines. Stabilization of soluble gp140 (sgp140) Env trimers has been achieved by introduction of an SOS disulfide bond linking gp120 and gp41, substitution of proline for isoleucine 559, and truncation of the gp41 ectodomain at residue 664. Crystal and cryo-EM structures of these sgp140 SOSIP.664 trimers have been solved. A cryo-EM structure of a cytoplasmic tail-deleted Env trimer (Env∆CT) in complex with the PGT151 neutralizing antibody was virtually identical to the sgp140 SOSIP.664 structure. Although these HIV-1 Env trimer structures are widely believed to represent the closed State-1 conformation, recent data from single- molecule Fluorescence Resonance Energy Transfer (smFRET) and crosslinking/mass spectrometry analyses indicate that this assumption is incorrect. In fact, the gp120 subunits of the sgp140 SOSIP.664 trimers and the Env∆CT/PGT151 complex are both in a State 2-like conformation! Crosslinking/mass spectrometry of the native cell-surface Env and sgp140 SOSIP.664 trimers revealed differences in the conformation of several gp120 and gp41 regions! The surprising results described above indicate that substantial differences exist between the conformations of the native State-1 Env and the structurally well-characterized sgp140 SOSIP.664 and PGT151-bound Env∆CT trimers, which apparently represent a default intermediate (State 2-like) conformation. Thus, we currently lack detailed information about the structure of the State-1 HIV-1 Env trimer, the major target for neutralizing antibodies and virus entry inhibitors. The proposed work seeks to devise approaches to express and purify HIV- 1 Env trimers enriched in a State-1 conformation. Given the importance of shape to the humoral immune system, we will then test the hypothesis that preparations of State 1-enriched immunogens will elicit primary HIV-1- neutralizing antibodies more efficiently than Envs not specifically enriched for a State-1 conformation.

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 ((gp120/gp41)3)。作为病毒表面唯一的病毒特异性分子，Env 是病毒攻击的主要靶标。 宿主中和抗体。在病毒进入过程中，与受体的结合会引发构象变化 亚稳态环境允许病毒和靶细胞膜融合。 Env 构象灵活性，即 对于病毒进入很重要，也有助于 HIV-1 逃避宿主抗体反应的能力。受体之前 接合，病毒粒子上的 HIV-1 Env 三聚体可以潜在地采样三种构象：亚稳态“封闭” 构象（状态 1）、“开放”CD4 结合构象（状态 3）和中间“部分开放” 构象（状态2）。原发性 HIV-1 包膜通过多个分子内和亚基间维持在状态 1 相互作用，使这些 Env 对潜在中和抗体的结合具有相对抵抗力。 CD4 结合将 Env 从状态 1 驱动到状态 2，然后进入状态 3，即前发夹中间体。 HIV-1 Env 三聚体的构象灵活性给结构研究和 疫苗的设计。通过引入可溶性 gp140 (sgp140) Env 三聚体已实现稳定化 连接 gp120 和 gp41 的 SOS 二硫键，用脯氨酸取代异亮氨酸 559，以及截短 gp41 胞外域位于残基 664。这些 sgp140 SOSIP.664 三聚体的晶体和冷冻电镜结构已 解决了。与 PGT151 复合的细胞质尾部缺失的 Env 三聚体 (EnvΔCT) 的冷冻电镜结构 中和抗体实际上与 sgp140 SOSIP.664 结构相同。虽然这些 HIV-1 包膜 人们普遍认为三聚体结构代表封闭的 State-1 构象，最近来自单聚体的数据 分子荧光共振能量转移 (smFRET) 和交联/质谱分析 表明这个假设是不正确的。事实上，sgp140 SOSIP.664 三聚体的 gp120 亚基和 EnvΔCT/PGT151 复合物均处于类似状态 2 的构象！天然物的交联/质谱分析 细胞表面 Env 和 sgp140 SOSIP.664 三聚体揭示了几种 gp120 和 sgp140 构象的差异 gp41地区！ 上述令人惊讶的结果表明构象之间存在显着差异 天然 State-1 Env 和结构良好表征的 sgp140 SOSIP.664 和 PGT151 结合的 EnvΔCT 三聚体，它显然代表默认的中间（类似状态 2）构象。因此，我们目前缺乏 有关 State-1 HIV-1 Env 三聚体结构的详细信息，该三聚体是中和的主要目标 抗体和病毒进入抑制剂。拟议的工作旨在设计表达和纯化艾滋病毒的方法 1 Env 三聚体以 State-1 构象富集。鉴于形状对体液免疫系统的重要性， 然后，我们将检验以下假设：富含状态 1 的免疫原制剂将引发原发性 HIV-1- 中和抗体比未专门富集 State-1 构象的 Env 更有效。