Impacts of Adaptive Coronavirus Evolution on Viral Membrane Fusion

项目摘要 SARS-CoV-2的持续适应产生了关注变体（VOC）。新的VOC出现 独立地和超越以前的，与不断增加的病毒适应性一致。大多数VOC 适应性存在于刺突（S）中，刺突是将病毒与细胞结合的复杂多结构域糖蛋白三聚体 并介导病毒-细胞膜融合。S受体结合结构域的充分表征适应 RBD改变RBD结构动力学、受体亲和力和抗体相互作用。然而，适应 其他S结构域在很大程度上仍被低估。这包括执行病毒-cell 膜融合该建议旨在阐明S2域中VOC适应性变化的后果。 目前的VOC，omicron（o），已经积累了六个S2突变。我们的主要目标是评估 这些和其他S2适应改变S蛋白动力学以促进人类细胞进入的方式。我们将利用 收集病毒膜融合和细胞进入测定系统，以确定S2变化是否重置了 瞬时S蛋白中间状态的动力学。这些过渡状态包括RBD升高， 从融合前到融合中间体再到融合后的受体结合和S2重折叠 配置.我们的检测系统将采用融合能力的病毒样颗粒（VLP）进行敏感性检测。 评估进入步骤，可溶性受体作为RBD暴露的探针，用于检测 特异性S过渡中间体，用于选择HR 2肽抗性的可复制VSVSARS-CoV-2-S 变异体、SARS-CoV-2复制子和重组SARS-CoV-2病毒， S2氨基酸取代导致病毒-细胞进入途径的重定向。 我们的初步结果表明，omicron S2适应性变构作用改变了S-受体 交互.第一个目标将确定S2融合结构域如何控制RBD及其相互作用 与受体。我们将确定控制受体反应性和S稳定性的特定VOC S2突变。我们 将决定这些突变如何改变病毒的进入要求。我们的初步结果还表明， omicron S2突变改变构象转变的速度，促进膜融合。的 第二个目标将确定S2突变如何控制融合结构域的动力学。我们将确定 S2结构转变的速率是否在VOC之间变化。我们还将选择和表征变体 对S2结构动力学的抑制剂具有抗性，并将发现这些抗性变体是否具有 对决定细胞对感染易感性的受体和蛋白酶的独特要求。 这项研究的结果将澄清目前模糊的选择性力量驱动人类适应过去（α， β、γ、δ）、当前（o）和未来VOC。我们希望这些结果能阐明膜融合的特性- 以揭示抑制CoV进入的新靶点的方式诱导S2结构域。

PROJECT SUMMARY The continuous adaptation of SARS-CoV-2 generates variants of concern (VOC). New VOCs arise independently and outcompete previous ones, consistent with ever increasing viral fitness. Most VOC adaptations reside in the spikes (S), the complex multidomain glycoprotein trimers that bind viruses to cells and mediate virus-cell membrane fusion. Well-characterized adaptations in the S receptor binding domains (RBDs) alter RBD structural dynamics, receptor affinities, and antibody interactions. However, adaptations in other S domains remain largely under-evaluated. This includes the S2 domains that execute virus-cell membrane fusion. This proposal aims to elucidate consequences of VOC adaptive changes in the S2 domains. The current VOC, omicron (o), has accumulated over six S2 mutations. Our central objective is to evaluate the ways these and other S2 adaptations alter S protein dynamics to facilitate human cell entry. We will utilize a collection of viral membrane fusion and cell entry assay systems to determine whether S2 changes reset the dynamics of transient S protein intermediate states. These transitory states include RBD elevations that control receptor binding and S2 refoldings from prefusion to fusion intermediate and through to postfusion configurations. Our assay systems will employ fusion-competent virus-like particles (VLPs) for sensitive evaluation of entry steps, soluble receptors as probes for RBD exposures, HR2 peptides for detection of specific S transitional intermediates, replication-competent VSVSARS-CoV-2-S for selecting HR2 peptide-resistant variants, and SARS-CoV-2 replicons and recombinant SARS-CoV-2 viruses for convincingly assigning specific S2 amino acid substitutions to consequential redirection of virus-cell entry pathways. Our preliminary results suggest that omicron S2 adaptations operate allosterically to alter S-receptor interactions. The first aim will determine how S2 fusion domains control RBDs and their interactions with receptors. We will identify specific VOC S2 mutations that control receptor reactivity and S stability. We will determine how these mutations change viral entry requirements. Our initial results also suggest that omicron S2 mutations change the pace of the conformational transitions facilitating membrane fusion. The second aim will determine how S2 mutations control dynamics of fusion domains. We will determine whether the rates of S2 structural transitions vary between VOCs. We will also select and characterize variants resistant to inhibitors of the S2 structural dynamics and will find out whether these resistant variants have unique requirements for the receptors and proteases that determine cell susceptibility to infection. The results of this study will clarify currently obscure selective forces driving the human adaptation of past (α, β, γ, δ), current (ο), and future VOCs. We expect the results will illuminate properties of the membrane fusion – inducing S2 domains in ways that reveal new targets for inhibition of CoV entry.