Probing conformational dynamics of HIV-1 Env in real time and in situ during virus entry

研究摘要：包膜（Env）糖蛋白是暴露在HIV表面的病毒编码蛋白， 1病毒粒子，是针对HIV-1的中和抗体的主要靶标。Env结构灵活， 构象动态，允许HIV-1通过膜融合进入易感细胞并逃避抗体 识别.因此，高度动态的Env是开发HIV-1疫苗、抑制剂和治疗的关键靶标 战略布局然而，缺乏对Env结构动力学的了解， 理解Env使病毒进入并促进免疫逃避的机制，这是在 环境导向干预的核心。Env是一种三聚体，其中每个原聚体由两个亚基gp 120和gp 125组成。 gp41。表面暴露的gp 120含有受体/辅助受体结合位点，跨膜gp 41 藏匿着核聚变机器当与受体CD 4结合时，gp 120经历构象变化， 导致辅受体结合位点的暴露。随后与辅助受体的相互作用将激活 gp 41中促进融合的级联重折叠事件，从亚稳定的融合前状态， 假定的前发夹中间体，到稳定的融合后六螺旋束，导致病毒的融合， 和细胞膜。Env的高分辨率结构极大地促进了我们对Env的理解 结构和抗原性。然而，Env的实时构象动力学， 描述了构成结构转换和熔合转换基础的事件序列 仍然很难找到单分子Förster共振能量转移（smFRET）应用于 将FRET配对荧光团引入gp 120以揭示病毒相关全长的动态特征 Env.我们已经证明，天然Env是动态的，并且在与CD 4相互作用后，从前Env转变为CD 4。 触发构象（状态1）通过部分打开中间（状态2）到完全激活打开 构象（状态3）。许多广泛中和抗体（bNAb）表现出对状态1-Env的偏好。 出乎意料的是，设计的可溶性Env、洗涤剂溶解的Env和醛硫醇-2灭活的病毒- 相关的Env都表现出占据状态2的倾向，提供了对电流谱的不同视角 基于Env的疫苗设计。我们使用smFRET对gp 120的研究为我们的下一个目标奠定了基础， 研究了解较少的融合蛋白gp 41。具体来说，我们建议使用动态和静态- smFRET和冷冻电子断层扫描（cryoET）的原位成像平台，以直接实时可视化 与膜融合相关并表征病毒包埋的gp 41的构象动力学/事件 结构上潜在的药物靶向GP 41中间体。最终目标是生成一个融合电影服务 作为发展HIV-1干预措施的框架，直接防止病毒进入。我们还将利用我们的平台 为gp 41定向中和的病毒相关Env指定构象识别偏好 抗体的研究，为抗体混合物和“抗体-疫苗”战略的发展提供信息。

Research Abstract: The envelope (Env) glycoprotein is a virus-encoded protein exposed on the surface of HIV- 1 virions and is the primary target of neutralizing antibodies against HIV-1. Env is structurally flexible or conformationally dynamic, allowing HIV-1 to enter susceptible cells via membrane fusion and evade antibody recognition. Thus, the highly dynamic Env is a critical target for developing HIV-1 vaccines, inhibitors, and cure strategies. However, a lack of knowledge on the structural dynamics of Env entangles the attempts to comprehend the mechanisms by which Env enables virus entry and facilitates immune evasion, which is at the core of Env-directed interventions. Env is a trimer in which each protomer consists of two subunits, gp120 and gp41. The surface-exposed gp120 contains receptor/coreceptor binding sites, and the transmembrane gp41 harbors the fusion machinery. Upon binding to the receptor CD4, gp120 undergoes conformational changes that lead to the exposure of a coreceptor-binding site. Subsequent interactions with the coreceptors will then activate a cascade of fusion-promoting refolding events in gp41, progressing from a metastable pre-fusion state, through the putative pre-hairpin intermediates, to the stable post-fusion six-helix bundle that leads to the fusion of viral and cellular membranes. High-resolution structures of Env have significantly furthered our understanding of Env architectures and antigenicity during virus entry. However, real-time conformational dynamics of Env that delineate the sequence of events that underlies the structural transformations and the fusogenic transitions remain largely elusive. Single-molecule Förster resonance energy transfer (smFRET) was applied upon introducing FRET-paired fluorophores into gp120 to reveal the dynamic features of virus-associated full-length Env. We have demonstrated that native Env is dynamic and, upon interaction with CD4, transitions from the pre- triggered conformation (State 1) through a partially open intermediate (State 2) to the fully activated open conformation (State 3). Many broadly neutralizing antibodies (bNAbs) demonstrate a preference for State 1-Env. Unexpectedly, the designed soluble Env, the detergent-solubilized Env, and the aldrithiol-2 inactivated virus- associated Env all exhibit a propensity to occupy State 2, offering a different perspective on the current spectra of Env-based vaccine design. Our studies on gp120 using smFRET have set the stage for our next goal—to investigate the less-understood fusion protein gp41. Specifically, we propose to use the dynamic-and-static in- situ imaging platform of smFRET and cryo-electron tomography (cryoET) to directly visualize real-time conformational dynamics/events of virus-embedded gp41 that correlate with membrane fusion and characterize potential drug-targeting gp41 intermediates structurally. The ultimate goal is to generate a fusion movie serving as the framework for developing HIV-1 interventions that direct against virus entry. We will also use our platform to specify conformational recognition preferences for virus-associated Env of gp41-directed neutralizing antibodies to inform the development of antibody cocktails and the “antibody-to-vaccine” strategy.