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

研究摘要：包膜糖蛋白是暴露在HIV病毒表面的一种病毒编码蛋白。 1病毒粒子，是针对HIV-1的中和抗体的主要目标。环境在结构上是灵活的或 构象动态，允许HIV-1通过膜融合进入敏感细胞并逃避抗体 承认。因此，高度动态的env是开发hiv-1疫苗、抑制剂和治愈药物的关键目标。 战略。然而，对环境的结构动力学缺乏了解，阻碍了试图 了解环境病毒使病毒进入和促进免疫逃避的机制，这是在 环境导向干预的核心。Env是一个三聚体，其中每个原型由两个亚基gp120和gp120组成。 Gp41。表面暴露的gp120含有受体/辅助受体结合部位，而跨膜gp41 隐藏着核聚变机器。Gp120与受体cd4结合后，会发生构象变化， 导致共受体结合部位的暴露。随后与辅助受体的相互作用将被激活 Gp41中一系列促进融合的折叠事件，从亚稳定的融合前状态发展到 推测的前发夹中间体，到导致病毒融合的稳定的融合后六螺旋束 和细胞膜。Env的高分辨率结构极大地加深了我们对Env的理解 病毒进入过程中的结构和抗原性。然而，环境的实时构象动力学 描绘了作为结构转换和融合转换基础的一系列事件 在很大程度上仍然难以捉摸。将单分子Förster共振能量转移(SmFRET)应用于 将FRET成对的荧光团引入gp120以揭示病毒相关全长的动态特征 环境我们已经演示了原生环境是动态的，并且在与CD4相互作用时，从之前的 通过部分开放的中间体(状态2)到完全激活的开放的触发构象(状态1 构象(状态3)。许多广谱中和抗体(BNAbs)表现出对State 1-Env的偏好。 出乎意料的是，设计的可溶性Env、洗涤剂增溶的Env和Aldrithiol-2灭活病毒- 相关的环境都显示出占据状态2的倾向，提供了对当前频谱的不同视角 基于环境的疫苗设计。我们使用smFRET对gp120的研究已经为我们的下一个目标奠定了基础 研究鲜为人知的融合蛋白gp41。具体地说，我们建议在- SMFRET和低温电子层析成像(CryoET)直接实时可视化的原位成像平台 与膜融合相关的病毒包埋gp41的构象动力学/事件及其特征 潜在的药物靶向gp41中间体的结构。最终目标是产生一部融合的电影服务 作为开发针对病毒入侵的艾滋病毒-1干预措施的框架。我们还将使用我们的平台 为gp41定向中和的病毒相关环境指定构象识别首选项 为开发抗体鸡尾酒和“抗体对疫苗”战略提供信息。

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.