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: 10425409
• 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/10425409
• 关键词: Acquired Immunodeficiency Syndrome; Amber; Antigens; Binding; CD4 Antigens; Cell membrane; Cells; Cellular Membrane; 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; vaccine-induced antibodies

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.

用平行smFRET和CryoET研究HIV-1包膜蛋白的结构和功能 摘要 HIV-1包膜蛋白通过促进病毒与细胞之间的融合而介导病毒进入细胞 膜。作为病毒颗粒表面唯一的病毒蛋白，HIV-1 Env也是 疫苗。疫苗开发的最大障碍是高度的序列变异性 Env，其致密的多糖屏蔽层和构象动力学。我们已经应用smFRET成像来获得洞察力 的构象状态和构象动力学的各个环境原 完整病毒粒子表面的天然三聚体。我们的工作揭示了环境从预先触发的 构象(状态1)通过必要的中间体(状态2)，形成一个与三个CD4受体结合的 构象(状态3)。许多由少数患者产生的广谱中和抗体(BNAbs)被发现 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关联 结构，我们做出了令人惊讶的观察，现存的高分辨率结构所基于的结构 均以占据下游的州2构象为主。因此，它的高分辨率结构 预触发态1Env，病毒表面的主要构象状态Env，即 优先被大多数广谱中和抗体(BNAbs)识别，从而识别中心疫苗 重要性--仍不得而知。为了验证和探索这些出乎意料的发现，我们建议将 SMFRET与其他结构方法相结合，以全面了解结构和动力学 在完整的病毒粒子表面的天然三聚体。为此，我们建立了紧密的联系 我们现有的Moths/Blanchard团队与刘军博士的实验室合作，以确定 病毒粒子表面不同状态(包括状态1)的HIV-1环境三聚体的结构 电子断层摄影术(冷冻电子显微镜)。利用smFRET和CryoET的内在协同效应，我们将捕获和 确定膜融合过程中病毒进入的结构中间体。