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

总结 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感染。 抗体的HIV-1 Env是病毒颗粒表面唯一的病毒蛋白，是疫苗的主要靶点 发展我们通过建立单分子Förster共振能量， 转移（smFRET），以监测在Env三聚体表面上的单个原聚体的取样。 病毒颗粒。smFRET数据表明，HIV-1 Env是构象动态的，并且样品至少三个 构象状态将观察到的FRET状态与现有的HIV-1 Env结构信息相关联 揭示了病毒表面的一种构象状态，这种状态无法用目前的高分辨率图像来解释。 结构.围绕smFRET数据的争议促使我们建立冷冻电子断层扫描 （cryoET）作为正交结构方法。cryoET和smFRET的优点是这两种方法都允许 用于表征病毒表面Env的结构和动力学。此外，我们还发现， 在进入过程的各个阶段阻止大量Env三聚体的实验条件， 通过cryoET和随后的亚断层图像平均进行结构表征。我们成功地建立了一个系统 我们将天然HIV-1病毒与携带受体CD 4和辅助受体的病毒样颗粒（VLP）一起孵育， 分子。我们观察到Env-CD 4 相对 CD4 膜 不对称 构象 CD4 的 可用性 在病毒表面，无法用目前的高分辨率结构来解释。 成品 states.对HIV-1 Env蛋白的结构和动力学的详细了解对于HIV-1的研究是重要的。 复合物聚集在膜-膜界面， 膜靠得更近。亚断层图像平均和分类显示，Env绑定一个 当膜进一步分开时，分子，然后接合两个，最后三个CD 4分子， 靠得更近了HIV-1 Env三聚体与一个和两个CD 4分子结合 构象状态这些cryoET研究回顾了我们实验室早期的smFRET工作， 在Env三聚体的开口中的中间体对应于具有单个不对称三聚体。 绑定在本提案中，我们将应用我们的cryoET方法来下一步对转变进行结构表征 Env从CD 4到辅助受体，然后活化成前发夹中间体。重新注入活力， 的cryoET和smFRET的进展，我们将继续努力，以表征一个 最后，我们建议 smFRET方法，以满足对不同Env构象的日益复杂的结构见解 构象状态 开发针对Env的疫苗和小分子抑制剂的免疫原。

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.