HIV-1 Env structure and function assessed by parallel smFRET and cryoET

通过平行 smFRET 和 CryoET 评估 HIV-1 Env 结构和功能

• 批准号: 9978713
• 负责人: Scott C Blanchard
• 金额: $ 83.81万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-18 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978713
• 关键词: Acquired Immunodeficiency Syndrome; Amber; Antigens; Binding; CD4 Antigens; Cell membrane; Cells; Cellular Membrane; Cleaved cell; Clinical Trials; Collaborations; Communities; Cryo-electron tomography; Event; Exhibits; Fluorescence Resonance Energy Transfer; Generations; Goals; HIV Envelope Protein gp120; HIV-1; Image; In Situ; Individual; Investigation; Laboratories; Ligands; Lipids; Mediating; Membrane; Membrane Fusion; Methods; Molecular Conformation; Monitor; Moths; Mutation; Patients; Polysaccharides; Property; Proteins; Protomer; Publishing; Reaction; Recombinants; Resolution; Site; Son of Sevenless Proteins; Structure; Surface; Technology; Tomogram; Transfer RNA; Vaccines; Vesicle; Viral; Viral Proteins; Virion; Virus; Work; base; design; disulfide bond; env Gene Products; fluorophore; insight; instrumentation; nanometer resolution; neutralizing antibody; preference; protein structure function; receptor; receptor binding; response; single molecule; synergism; unnatural amino acids; vaccine development

HIV-1 Env protein structure and function assessed by parallel smFRET and cryoET Summary The HIV-1 Env protein mediates virus entry into cells by promoting fusion between viral and cellular membranes. As the only virus protein on the surface of virus particles, HIV-1 Env is also a major target for vaccines. The greatest barriers to the development of a vaccine have been the high sequence variability of Env, its dense glycan shield and conformational dynamics. We have applied smFRET imaging to gain insights into the conformational states and conformational dynamics of individual Env protomers in the context of the native trimer on the surface of intact virions. Our work has revealed that Env opens from a pre-triggered conformation (State 1) through a necessary intermediate (State 2), into a three CD4 receptor-bound conformation (State 3). Many broadly neutralizing antibodies (bNAbs) generated by few patients were found to exhibit a preference for State 1. Associating these conformational states with existing high-resolution structures, we made the surprising observation that the constructs upon which extant high-resolution structures are based predominantly occupy the downstream State 2 conformation. Hence, the high-resolution structure of the pre-triggered State 1 Env, the predominant conformational state of Env on the surface of viruses, which is preferentially recognized by most broadly neutralizing antibodies (bNAbs) - and thus of central vaccine importance - remains unknown. To validate and explore these unexpected findings we propose to combine smFRET with other structural methods to gain a comprehensive understanding of the structure and dynamics of the native trimer on the surface of intact virions. Towards this end we have established closely knit collaborations between our existing Mothes/Blanchard team and the laboratory of Dr. Jun Liu to determine the structure of the HIV-1 Env trimer in its distinct states, including State 1, on the surface of virions by cryo- electron tomography (cryoET). Leveraging the inherent synergies of smFRET and cryoET, we will capture and determine structural intermediates of viral entry during membrane fusion.

通过平行smFRET和cryoET评估HIV-1 Env蛋白的结构和功能 总结 HIV-1 Env蛋白通过促进病毒与细胞融合介导病毒进入细胞 膜。作为病毒颗粒表面唯一的病毒蛋白，HIV-1 Env也是HIV-1感染的主要靶点。 疫苗。疫苗开发的最大障碍是免疫球蛋白的高序列变异性。 Env，其致密聚糖盾和构象动力学。我们应用smFRET成像来获得 的背景下，单个Env原聚体的构象状态和构象动力学。 完整病毒体表面的天然三聚体。我们的研究表明，Env是从一个预先触发的 构象（状态1）通过必要的中间体（状态2），转化为三个CD 4受体结合的 构象（状态3）。发现少数患者产生的许多广泛中和抗体（bNAb） 表现出对状态1的偏好。将这些构象状态与现有的高分辨率 结构，我们做出了令人惊讶的观察，即现存高分辨率结构的构建体 主要占据下游状态2构象。因此， 预触发状态1 Env，Env在病毒表面的主要构象状态， 优先被大多数广泛中和抗体（bNAb）识别-因此也是中央疫苗的识别 重要性-仍然未知。为了验证和探索这些意想不到的发现，我们建议将联合收割机 smFRET与其他结构方法，以获得结构和动力学的全面了解 在完整病毒体表面的天然三聚体。为此，我们建立了紧密的联系。 我们现有的Mothes/Blanchard团队和Jun Liu博士的实验室之间的合作， 通过低温冷冻，在病毒体表面上以其不同状态（包括状态1）的HIV-1 Env三聚体的结构， 电子断层扫描（cryoET）。利用smFRET和cryoET的固有协同作用，我们将捕获和 确定膜融合过程中病毒进入的结构中间体。