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

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

• 批准号: 10761955
• 负责人: WALTHER H MOTHES
• 金额: $ 82.95万
• 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 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10761955
• 关键词: Adopted; Affect; Antibodies; Antigens; Antiviral Therapy; Automobile Driving; Binding; Binding Proteins; Bulla; CCR5 gene; CD4 Antigens; Cell membrane; Cells; Cellular Membrane; Chimeric Proteins; Cholesterol; Chronic; Classification; Complex; Cryo-electron tomography; Data; Development; Environment; Fluorescence Resonance Energy Transfer; HIV; HIV Envelope Protein gp120; HIV-1; Image; Incubated; Laboratories; Lipids; Mediating; Membrane; Membrane Fusion; Methodology; Methods; Molecular Conformation; Monitor; Positioning Attribute; Process; Protein Conformation; Protein Dynamics; Proteins; Protomer; Receptor Activation; Resolution; Sampling; Speed; Structure; Surface; System; T-Lymphocyte; Testing; Vaccine Design; Vaccines; Viral; Viral Proteins; Virion; Virus; Virus-like particle; Visualization; Work; data exchange; env Gene Products; fluorophore; insight; instrumentation; particle; protein function; protein structure; receptor; single molecule; small molecule inhibitor; software development; therapy design; vaccine development; Adopted; Affect; Antibodies; Antigens; Antiviral Therapy; Automobile Driving; Binding; Binding Proteins; Bulla; CCR5 gene; CD4 Antigens; Cell membrane; Cells; Cellular Membrane; Chimeric Proteins; Cholesterol; Chronic; Classification; Complex; Cryo-electron tomography; Data; Development; Environment; Fluorescence Resonance Energy Transfer; HIV; HIV Envelope Protein gp120; HIV-1; Image; Incubated; Laboratories; Lipids; Mediating; Membrane; Membrane Fusion; Methodology; Methods; Molecular Conformation; Monitor; Positioning Attribute; Process; Protein Conformation; Protein Dynamics; Proteins; Protomer; Receptor Activation; Resolution; Sampling; Speed; Structure; Surface; System; T-Lymphocyte; Testing; Vaccine Design; Vaccines; Viral; Viral Proteins; Virion; Virus; Virus-like particle; Visualization; Work; data exchange; env Gene Products; fluorophore; insight; instrumentation; particle; protein function; protein structure; receptor; single molecule; small molecule inhibitor; software development; therapy design; vaccine development

Summary The HIV-1 envelope protein (Env) mediates virus entry into cells while protecting functional centers from antibodies. As the only virus protein on the surface of virus particles, HIV-1 Env is a major target for vaccine development. We initiated studies on HIV-1 Env by establishing single-molecule Förster Resonance Energy Transfer (smFRET) to monitor the sampling of a single protomer in the context of a Env trimer on the surface of a virus particle. smFRET data indicated that HIV-1 Env is conformationally dynamic and samples at least three conformational states. Associating the observed FRET states with existing HIV-1 Env structural information revealed a conformational state on the surface of viruses that cannot be explained by current high-resolution structures. The controversy surrounding smFRET data prompted us to establish cryo-electron tomography (cryoET) as an orthogonal structural method. The advantage of cryoET and smFRET is that both methods allow for characterization of the structure and dynamics of Env on the surface of viruses. In addition, we identified experimental conditions that arrest a high number of Env trimers at various stages of the entry process to allow a structural characterization by cryoET and subsequent subtomogram averaging. We succeeded with a system where we incubate native HIV-1 viruses with virus-like particles (VLP) carrying receptor CD4 and coreceptor molecules. We observed Env-CD4 opposing CD4 membranes asymmetric conformational CD4 of availability on the surface of viruses that cannot be explained by current high-resolution structures. refined states. A detailed understanding of the structure and dynamics of the HIV-1 Env protein is important for the complexes to cluster at membrane-membrane interfaces thereby bringing membranes closer together. Subtomogram averaging and classification revealed that Env bound one molecule when membranes were further apart, then engaged two, and finally three CD4 molecules as moved closer together. HIV-1 Env trimers bound to one and two CD4 molecules adopted conformational states. These cryoET studies recall earlier smFRET work in our laboratory that a intermediate in the opening of the Env trimer corresponds to an asymmetric trimer with a single bound. I n this proposal, we will apply our cryoET methodology to next structurally characterize the transition Env from CD4 to coreceptor followed by activation into the pre-hairpin intermediate. Reenergized by the of cryoET and advances on smFRET, we will pursue our efforts to characterize a Finally, we are proposing smFRET methods to meet the increasingly complex structural insights into distinct Env conformational conformational state development of immunogens for vaccines and small molecule inhibitors against Env.

概括 HIV-1包膜蛋白（ENV）介导病毒进入细胞，同时保护功能中心免受 抗体。作为病毒颗粒表面上唯一的病毒蛋白，HIV-1 Env是疫苗的主要靶标 发展。我们通过建立单分子förster共振能量开始了HIV-1 Env的研究 转移（SMFRET）以监视在env trimer表面的环境中的单个杂物的采样 病毒粒子。 SMFRET数据表明HIV-1 Env在构象上是动态的，样品至少三个 构象状态。将观察到的粮食状态与现有的HIV-1环境结构信息相关联 揭示了病毒表面上无法通过当前高分辨率解释的构象状态 结构。围绕SMFRET数据的争议促使我们建立了冷冻电子断层扫描 （冷冻）作为正交结构方法。冷冻和SMFRET的优点是，这两种方法都允许 为了表征病毒表面上的结构和动力学。另外，我们确定了 实验条件在进入过程的各个阶段都会阻止大量的ENV三聚体以允许 冷冻和随后的子图平均图的结构表征。我们成功了一个系统 在其中我们将携带受体CD4和共感染者的病毒样颗粒（VLP）孵育的天然HIV-1病毒孵育 分子。我们观察到env-cd4 反对 CD4 膜 不对称 构象 CD4 的 可用性 在病毒的表面上无法通过当前的高分辨率结构来解释。 精制 国家。对HIV-1 Env蛋白的结构和动态的详细理解对于 复合物以聚集在膜膜界面上，从而带来 膜更近。亚图平均图和分类表明，env绑定了一个 当膜进一步分开时，分子，然后参与两个，最后三个CD4分子作为 近距离移动。 HIV-1 ENV夹子结合到一个和两个CD4分子 构象状态。这些冷冻研究回顾了我们实验室中早期的SMFRET工作， Env触发器开口的中间体对应于一个不对称触发器 边界。在此提案中，我们将使用冷冻方法来在结构上进行下一个结构表征过渡 从CD4到共核能，然后将其激活到发行前中间体中。被 冷冻和SMFRET的进步，我们将努力以表征 最后，我们正在提议 SMFRET方法可以满足日益复杂的结构洞察到不同的Env构象 构象状态 开发针对Env的疫苗和小分子抑制剂的免疫原子。