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)三聚体((gp120/gp41)3)。作为病毒表面唯一的病毒特异性分子，env是 宿主中和抗体。在病毒进入过程中，与受体的结合触发了 亚稳的Env使病毒和靶细胞膜融合。环境构象灵活性，这是 重要的是病毒进入，也有助于艾滋病毒-1‘S逃避宿主抗体反应的能力。先于受体 通过接触，病毒粒子上的HIV-1环境三聚体可能会采样三种构象：亚稳的“封闭”构象 构象(状态1)、“开放”的结合CD4的构象(状态3)和中间的“部分开放”构象 构象(状态2)。主要HIV-1环境蛋白由多个分子内和亚基间维持在状态1 相互作用，使这些环境相对抵抗潜在的中和抗体的结合。CD_4 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环境三聚体的构象灵活性给结构研究和 疫苗的设计。可溶性gp140(Sgp140)环境三聚体的稳定是通过引入 连接gp120和gp41的SOS二硫键，用Pro取代异亮氨酸559，以及截断 Gp41胞外区，残基664位。这些sgp140 SOSIP.664三聚体的晶体结构和低温电子显微镜结构 解决了。胞质尾部缺失环状三聚体(Env∆CT)与PGT151复合体的低温电子显微镜结构 中和抗体与sgp140 SOSIP.664结构基本相同。尽管这些HIV-1环境 三聚体结构被广泛认为代表闭合状态-1构象，最近来自单一-1的数据- 分子荧光共振能量转移(SmFRET)和交联/质谱分析 表明这一假设是不正确的。事实上，sgp140 SOSIP.664三聚体的gp120亚基和 Env∆CT/PGT151复合体均为类状态2构象！天然产物的交联/质谱学 细胞表面环境和sgp140 SOSIP.664三聚体显示了几种gp120和sgp140 SOSIP.664三聚体的构象差异 Gp41区域！ 上面描述的令人惊讶的结果表明，在构象之间存在着显著的差异。 天然状态-1环境和结构特征良好的sgp140 SOSIP.664和PGT151结合的环境∆CT 三聚体，这显然代表了默认的中间体(类状态2)构象。因此，我们目前缺乏 关于国家-1艾滋病毒-1环境三聚体的结构的详细信息，这是中和的主要目标 抗体和病毒进入抑制剂。这项拟议的工作旨在设计出表达和纯化艾滋病毒的方法- 1个富含State-1构象的环境三聚体。鉴于形状对体液免疫系统的重要性， 然后，我们将测试这一假设，即浓缩状态1的免疫原制剂将诱导原发HIV-1- 对于State-1构象，中和抗体比非特定浓缩的Envs更有效。