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

在病毒进入过程中实时原位探测 HIV-1 Env 的构象动力学

基本信息

批准号：
10660408
负责人：
Maolin Lu
金额：
$ 37.07万
依托单位：
UNIVERSITY OF TEXAS HLTH CTR AT TYLER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10660408
关键词：
Antibodies Antigens Architecture Binding Binding Sites CD4 Antigens Cells Cellular Membrane Chemistry Chimeric Proteins Cryo-electron tomography Detergents Development Drug Targeting Dyes Event Evolution Exhibits Exposure to Fluorescence Resonance Energy Transfer Genetic Code Glycoproteins Goals HIV HIV Envelope Protein gp120 HIV-1 Immune Immune Evasion In Situ Intervention Knowledge Lead Length Light Membrane Membrane Fusion Molecular Molecular Conformation Monitor Peptides Polysaccharides Proteins Protomer Research Resolution Specific qualifier value Structure Surface T-20 Technology Testing Therapeutic Therapeutic Intervention Time To specify Vaccine Design Vaccines Viral Virion Virus War Work base conformational conversion design drug development env Gene Products flexibility fluorophore imaging platform in situ imaging inhibitor insight movie neutralizing antibody novel preference prophylactic rational design receptor receptor binding single molecule small molecule small molecule inhibitor vaccine development vaccine strategy

项目摘要

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.

研究摘要：包膜糖蛋白（Env）是暴露在HIV表面的病毒编码蛋白， 1 病毒粒子，是 HIV-1 中和抗体的主要靶标，Env 结构灵活或结构灵活。构象动态，使HIV-1能够通过膜融合进入易感细胞并逃避抗体因此，高度动态的 Env 是开发 HIV-1 疫苗、抑制剂和治疗方法的关键靶点。然而，缺乏对结构动力学的了解阻碍了尝试。了解 Env 使病毒进入并促进免疫逃避的机制，这是最重要的 Env 介导的干预措施的核心是一个三聚体，其中每个原聚体由两个亚基 gp120 和 gp120 组成。 gp41 表面暴露的 gp120 包含受体/辅助受体结合位点，跨膜 gp41。与受体 CD4 结合后，gp120 会发生构象变化。导致辅助受体结合位点的暴露，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重折叠事件，从亚稳态融合前状态发展到假定的前发夹中间体，到导致病毒融合的稳定的融合后六螺旋束 Env 和细胞膜的高分辨率结构极大地加深了我们对 Env 的理解。然而，Env 的实时构象动态。描述结构转变和融合转变背后的事件顺序单分子福斯特共振能量转移（smFRET）的应用仍然在很大程度上难以捉摸。将 FRET 配对荧光团引入 gp120，揭示病毒相关全长的动态特征我们已经证明，原生 Env 是动态的，并且在与 CD4 相互作用时，会从前环境转变为环境。触发构象（状态1）通过部分开放中间体（状态2）到完全激活开放许多广泛中和抗体 (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、去污剂溶解的Env和aldrithiol-2灭活病毒—— 相关的 Env 都表现出占据状态 2 的倾向，为当前光谱提供了不同的视角我们使用 smFRET 对 gp120 进行的基于 Env 的疫苗设计的研究为我们的下一个目标奠定了基础。具体来说，我们建议使用动态和静态的方法来研究不太了解的融合蛋白 gp41。 smFRET 和冷冻电子断层扫描 (cryoET) 的原位成像平台可直接实时可视化与膜融合相关的病毒嵌入 gp41 的构象动力学/事件并表征潜在的药物靶向 gp41 中间体的结构最终目标是生成融合电影。作为开发针对病毒进入的 HIV-1 干预措施的框架，我们还将使用我们的平台。指定 gp41 定向中和的病毒相关 Env 的构象识别偏好抗体为抗体鸡尾酒的开发和“抗体到疫苗”策略提供信息。