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

摘要 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。作为病毒颗粒表面唯一的病毒蛋白，HIV-1 env是疫苗的主要靶点 发展。我们通过建立单分子Förster共振能量启动了对HIV-1环境病毒的研究 转移(SmFRET)，用于在表面的环境三聚体的背景下监测单个原核的采样 一种病毒颗粒。SmFRET数据表明，HIV-1 env是构象动态的，至少有三个样本 构象状态。将观察到的FRET状态与现有的HIV-1环境结构信息相关联 揭示了病毒表面的一种构象状态，这是目前高分辨率无法解释的 结构。围绕smFRET数据的争论促使我们建立了冷冻电子断层扫描 (CryoET)作为一种正交结构方法。CryoET和smFRET优点在于这两种方法都允许 用于表征病毒表面包膜病毒的结构和动力学。此外，我们还确定了 在进入过程的不同阶段阻止大量环境三聚体的实验条件 用低温电子显微镜和随后的亚断层图像平均法进行结构表征。我们用一套系统取得了成功 我们将本地HIV-1病毒与携带受体CD4和辅助受体的病毒样颗粒(VLP)孵化 分子。我们观察了Env-CD4 对立的 CD_4 膜 不对称 构象 CD_4 的 可用性 目前的高分辨率结构无法解释的病毒表面。 精炼 各州。对HIV-1包膜蛋白的结构和动力学的详细了解对于 络合物聚集在膜-膜界面，从而带来 膜靠得更近了。亚断层图像平均和分类显示环境边界为1 当膜离得更远时，分子，然后接合两个，最后三个CD4分子作为 靠得更近了。通过与一个和两个CD4分子结合的HIV-1环境三聚体 构象状态。这些低温晶体管研究让人回想起我们实验室早期的smFRET工作， 环境三聚体开口中的中间体对应于具有单一 被绑住了。在这项提议中，我们将应用我们的冷冻ET方法来下一步从结构上表征这种转变 包膜蛋白从CD4到辅受体，然后激活成发夹前中间体。重新焕发了活力 的进展，我们将继续努力描述一种 最后，我们提议 满足对不同环境构象日益复杂的结构洞察的smFRET方法 构象状态 针对env的疫苗免疫原和小分子抑制剂的开发。

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.