Impacts of Adaptive Coronavirus Evolution on Viral Membrane Fusion

冠状病毒适应性进化对病毒膜融合的影响

• 批准号: 10727448
• 负责人: Thomas Miller Gallagher
• 金额: $ 24.48万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-14 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10727448
• 关键词: 2019-nCoV; Affinity; Amino Acid Substitution; Amino Acids; Animals; Antibodies; Apical; Binding; Biochemical; Biological; Biological Assay; Cell membrane; Cells; Collection; Communication; Communities; Complex; Coronavirus; Coronavirus spike protein; Detection; Distant; Endocytosis; Engineering; Equilibrium; Evaluation; Evolution; Future; Glycoproteins; Goals; Human; Infection; Kinetics; Link; Measures; Mediating; Membrane Fusion; Middle East Respiratory Syndrome Coronavirus; Molecular Conformation; Mutation; Pathway interactions; Peptide Hydrolases; Peptides; Predisposition; Property; Protein Conformation; Protein Dynamics; Proteins; Receptor Cell; Recombinants; Replicon; Resistance; SARS-CoV-2 B.1.1.529; SARS-CoV-2 spike protein; SARS-CoV-2 variant; Site; Structure; Surface; System; Variant; Viral; Viral Drug Resistance; Virulence; Virus; Virus Receptors; Virus Shedding; Virus-like particle; conformational conversion; driving force; global health; inhibitor; insight; receptor; receptor binding; recombinant virus; resistance mutation; tool; transmission process; variants of concern; viral fitness; virus tropism; 2019-nCoV; Affinity; Amino Acid Substitution; Amino Acids; Animals; Antibodies; Apical; Binding; Biochemical; Biological; Biological Assay; Cell membrane; Cells; Collection; Communication; Communities; Complex; Coronavirus; Coronavirus spike protein; Detection; Distant; Endocytosis; Engineering; Equilibrium; Evaluation; Evolution; Future; Glycoproteins; Goals; Human; Infection; Kinetics; Link; Measures; Mediating; Membrane Fusion; Middle East Respiratory Syndrome Coronavirus; Molecular Conformation; Mutation; Pathway interactions; Peptide Hydrolases; Peptides; Predisposition; Property; Protein Conformation; Protein Dynamics; Proteins; Receptor Cell; Recombinants; Replicon; Resistance; SARS-CoV-2 B.1.1.529; SARS-CoV-2 spike protein; SARS-CoV-2 variant; Site; Structure; Surface; System; Variant; Viral; Viral Drug Resistance; Virulence; Virus; Virus Receptors; Virus Shedding; Virus-like particle; conformational conversion; driving force; global health; inhibitor; insight; receptor; receptor binding; recombinant virus; resistance mutation; tool; transmission process; variants of concern; viral fitness; virus tropism

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.

项目摘要 SARS-COV-2的连续适应会产生关注的变体（VOC）。出现了新的VOC 独立和胜任以前的，与病毒性健身的增加一致。大多数VOC 适应性驻留在尖峰中，尖峰，复杂的多域糖蛋白三聚体，将病毒与细胞结合 并介导病毒细胞膜融合。 S受体结合结构域中的特征良好适应 （RBD）改变RBD结构动力学，受体亲和力和抗体相互作用。但是，适应 其他S域在很大程度上保持不足。这包括执行病毒细胞的S2域 膜融合。该建议旨在阐明S2域中VOC自适应变化的后果。 当前的VOC Omicron（O）已在六个S2突变上积累。我们的核心目标是评估 这些和其他S2适应改变了S蛋白动力学以促进人体细胞的进入。我们将利用一个 收集病毒膜融合和细胞进入测定系统，以确定S2是否改变重置 瞬态S蛋白中间状态的动力学。这些暂时状态包括控制的RBD高程 受体结合和从融合到中间的融合到融合的S2重新折叠 配置。我们的测定系统将员工融合能力的病毒样颗粒（VLP）敏感 评估入口步骤，实体受体作为RBD暴露问题，HR2肽检测 特定的S过渡中间体，具有复制能力的VSVSARS-COV-2-S，用于选择耐HR2胡椒粉 变体，SARS-COV-2复制品和重组SARS-COV-2病毒，可令人信服地分配特定 S2氨基酸取代病毒细胞进入途径。 我们的初步结果表明，Omicron S2适应作用以变构为改变S受体 互动。第一个目标将决定S2融合域如何控制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 将确定这些突变如何改变病毒进入要求。我们的最初结果也表明 Omicron S2突变改变了支撑膜融合的构象转变的速度。这 第二个目标将决定S2突变如何控制融合域的动力学。我们将确定 S2结构过渡的比率在VOC之间有所不同。我们还将选择并表征变体 对S2结构动力学的抑制剂具有抗性，并且会发现这些抗性变体是否具有 对于确定细胞感染敏感性的接收器和蛋白酶的独特要求。 这项研究的结果将阐明当前掩盖了推动人类过去适应的选择性的选择性（α， β，γ，δ），电流（ο）和未来的VOC。我们预计结果将阐明膜融合的特性 - 诱导S2域的方式揭示了抑制COV进入的新目标。