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.

项目摘要/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）Trimer（（GP120/GP41）3）。作为病毒表面上唯一的病毒特异性分子，env是 宿主中和抗体。在进入病毒期间，与接收器的结合触发了构象变化 允许病毒和靶细胞膜融合的亚稳态ENV。 env构象灵活性，这是 对于病毒的侵害很重要，也有助于HIV-1逃避宿主抗体反应的能力。接收器之前 参与性的HIV-1 env trimer对病毒粒子可能有可能采样三种构象：亚稳态的“封闭” 构象（状态1），“开放” CD4结合构象（状态3）和中间的“部分打开” 构象（状态2）。原代HIV-1 ENV通过多个分子内和亚基维持在状态1中 相互作用，使这些Envs对潜在中和抗体的结合相对抵抗力。 CD4 结合从状态1到状态2的驱动器驱动器，然后驱动到状态3，前hairpin中间体。 HIV-1 Env Trimers的构象灵活性为结构研究和 疫苗的设计。实心GP140（SGP140）ENV三聚体的稳定已通过引入 SOS二硫键连接GP120和GP41，脯氨酸代替异亮氨酸559，以及截断 居住在664的GP41胞外域。这些SGP140 SOSIP的晶体和冷冻EM结构已已 已解决。与PGT151的复合物中的细胞质尾部删除的Env Trimer（EnvΔCT）的冷冻EM结构 中和抗体几乎与SGP140 SOSIP.664结构相同。虽然这些HIV-1环境 人们普遍认为，三聚体结构代表了封闭状态-1的组成，来自单一的数据的最新数据 分子荧光共振能传递（SMFRET）和交联/质谱分析 实际上，SGP140 SOSIP的GP120亚基。664三聚机和 EnvΔCT/PGT151复合物都处于状态2样构象中！天然的交联/质谱法 细胞表面Env和SGP140 SOSIP.664三聚体揭示了几种GP120和 GP41区域！ 上述令人惊讶的结果表明，构象之间存在实质性差异 本地状态-1 env和结构良好的SGP140 SOSIP.664和PGT151-BONDENVΔCT 三聚体，显然代表了默认的中间（状态2状）构象。那我们目前缺乏 有关状态1 HIV-1 env触发结构的详细信息，这是中和的主要目标 抗体和病毒进入抑制剂。拟议的工作旨在设计表达和净化艾滋病毒的方法 1 ENV的三聚体富含状态1构象。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：富含州1富集的免疫原的制剂将引起原发性HIV-1-- 与未针对状态1构象特别丰富的ENV相比，中和抗体更有效。