The role of conformational dynamics of enveloped virus spike proteins in cell entry

研究摘要：存在于人类包膜病毒表面的刺突/融合糖蛋白是 免疫反应的靶点，是研究人员开发疫苗和抗病毒治疗的重点。的 刺突蛋白的构象动力学驱动包膜病毒通过病毒膜进入细胞 融合并促进抗体识别。然而，由于缺乏对动态的深入了解， 完整阐明刺突蛋白促进病毒进入的分子机制。很多病毒， 如冠状病毒SARS-CoV-2、呼吸道合胞病毒（RSV）和HIV-1，具有相似病毒融合 机制（I型）由各自的刺突蛋白介导。这些尖刺经历了戏剧性的结构变化 变化，并且从构象转变释放的能量克服了融合动力学障碍。 然而，我们对多步融合过程的理解主要依赖于单个结构快照， 融合终点的刺突蛋白。这些终点如何以时间分辨的方式相关，以及顺序 和病毒进入基础的构象事件的频率仍然很难确定。拟议研究 扩展了我们探索病毒膜融合的努力，并得到了我们探索病毒膜融合的经验的支持。 SARS-CoV-2刺突（S）和HIV-1包膜（Env）糖蛋白的构象动力学。总体 这个项目的目标是将我们的知识整合到一个通用的工作模型中，该模型将描述一个时间分辨的 I型融合机制的逐步框架，其中融合蛋白的构象轨迹 在空间和时间上都有明确的定义。我们率先使用单分子福斯特共振能量 转移（smFRET）研究S，并揭示了病毒上的多个S构象。我们描绘了 在细胞受体激活后，S从封闭构象转变为开放构象。我们提供了第一个 从开放状态减速过渡动力学的实验证据，表明增加的稳定性， 融合反应开放状态将成为SARS-CoV-2适应策略的一部分。在这里，我们将使用集成的 smFRET和病毒与细胞融合的平台与计算和结构工具相结合，以揭示 构象可塑性S在病毒进化过程中适应，并使构象轨迹S可视化 在融合过程中。我们将对另一种呼吸道病毒- RSV融合体（F）进行比较研究。 蛋白，与其他新出现的病毒的I型刺突蛋白的兴趣。我们将阐明构象 事件和F介导的病毒膜融合的转换动力学，并评估是否构象- 基于F的候选疫苗的呈递代表暴露于宿主的主要状态。研究 有望使我们能够识别S和F介导的融合过程的共同和不同特征 这将推进我们的知识，并帮助我们确定第一类聚变机制的共同主题。我们 我设想，这项使用不同先进技术的研究计划将揭示一些尚未被认识到的见解 根据NIGMS的使命，

Research Abstract: The spike/fusion glycoproteins residing on the surface of human enveloped viruses are targets of immune response and are the focus of researchers developing vaccines and antiviral treatments. The conformational dynamics of spike proteins drive the entry of enveloped viruses into cells via viral membrane fusion and facilitate antibody recognition. However, the lack of deep insights into dynamics has prevented a complete elucidation of the molecular mechanism by which spike proteins promote virus entry. Many viruses, such as coronavirus SARS-CoV-2, respiratory syncytial virus (RSV), and HIV-1, share a similar viral fusion mechanism (type-I) mediated by their respective spike proteins. These spikes undergo dramatic structural changes, and the energy released from conformational transitions overcomes the fusion kinetic barriers. However, our understanding of the multi-step fusion process mainly relies on individual structural snapshots of spike proteins at fusion endpoints. How these endpoints are correlated in a time-resolved manner and the order and frequency of conformational events underlying virus entry remain largely elusive. The proposed research extends our efforts to explore viral membrane fusion and is supported by our experience probing the conformational dynamics of the SARS-CoV-2 spike (S) and HIV-1 envelope (Env) glycoproteins. The overarching goal of this project is to integrate our knowledge into a generic working model that will describe a time-resolved stepwise framework of the type-I fusion mechanism, in which the conformational trajectories of fusion proteins are explicitly defined in space and time. We pioneered the use of the single-molecule Förster resonance energy transfer (smFRET) to study S and revealed multiple S conformations on the virus. We delineated sequential transitions of S from closed to open conformations upon activation by cellular receptors. We provided the first experimental evidence of decelerated transition dynamics from open states, suggesting increased stability of the fusion-reactive open state to be part of the SARS-CoV-2 adaption strategies. Here, we will use an integrated platform of smFRET and virus-to-cell fusion in combination with computational and structural tools to reveal the conformational plasticity S adapts during virus evolution and to visualize the conformational trajectory S undergoes during fusion. We will perform comparative studies on another respiratory virus - RSV fusion (F) protein, with interest in other type-I spike proteins of newly emerging viruses. We will elucidate conformational events and transition dynamics of F-mediated viral membrane fusion and evaluate whether conformation- presentation of F-based vaccine candidates represents the predominant state exposed to the host. The studies are expected to allow us to identify the common and divergent traits of the S- and F-mediated fusion processes that will advance our knowledge and help us define the common theme of the type-I fusion mechanism. We envision that this program of research using different advanced technologies will reveal unrecognized insights into virus entry that lay the foundation for advances in anti-viral interventions, in line with the mission of NIGMS.