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 是免疫反应的靶标，也是研究人员开发疫苗和抗病毒治疗的重点。这个 S蛋白的构象动力学驱动包膜病毒通过病毒膜进入细胞 融合和促进抗体识别。然而，对动力学缺乏深入的洞察，阻碍了 全面阐明尖峰蛋白促进病毒入侵的分子机制。很多病毒， 如冠状病毒SARS-CoV-2、呼吸道合胞病毒(RSV)和HIV-1共享类似病毒融合 机制(类型-I)由各自的SPEKE蛋白介导。这些尖峰经历了戏剧性的结构 变化，构象跃迁释放的能量克服了融合动力学势垒。 然而，我们对多步骤融合过程的理解主要依赖于单个的结构快照 融合终点处的尖峰蛋白。这些端点如何以时间分辨的方式进行关联以及顺序 病毒进入的基础构象事件的频率在很大程度上仍然难以捉摸。拟议的研究 扩展了我们探索病毒膜融合的努力，并得到了我们探索 SARS-CoV-2尖峰蛋白(S)和HIV-1包膜糖蛋白的构象动力学最重要的是 这个项目的目标是将我们的知识集成到一个通用的工作模型中，该模型将描述一个时间解析的 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。我们率先使用了单分子Förster共振能量 转移(SmFRET)对S进行研究，发现病毒上存在多个S构象。我们描绘了一个连续的 S被细胞受体激活后从封闭构象到开放构象的转变。我们提供了第一个 从开放状态减速转变动力学的实验证据，表明 将聚变反应开放状态作为SARS-CoV-2适应策略的一部分。在这里，我们将使用一个集成的 SmFRET和病毒到细胞融合的平台结合计算和结构工具揭示 S在病毒进化过程中的构象可塑性及其构象轨迹可视化 在融合过程中经历。我们将对另一种呼吸道病毒-RSV融合(F)进行比较研究 蛋白质，对新出现的病毒的其他I型尖峰蛋白感兴趣。我们将阐明构象 F-介导的病毒膜融合的事件和过渡动力学，并评估构象- 以F为基础的候选疫苗的呈现代表了接触到宿主的主要状态。这些研究 有望使我们识别S和F介导的融合过程的共同和不同特征 这将增进我们的知识，并帮助我们确定第一类聚变机制的共同主题。我们 设想这项使用不同先进技术的研究计划将揭示未被认可的见解 根据NIGMS的使命，为在抗病毒干预方面取得进展奠定了基础。

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.