Project 4: Molecular Mechanisms of RSV F Activation and Inhibition

项目4：RSV F激活和抑制的分子机制

• 批准号: 8813299
• 负责人: Jason Scott McLellan
• 金额: $ 26.78万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8813299
• 关键词: 1 year old; Acute; Address; Adopted; Antibodies; Antiviral Agents; Binding; Binding Sites; Cell Surface Receptors; Cell surface; Cells; Cellular Membrane; Centers of Research Excellence; Cessation of life; Child; Collection; Complex; Data Engineering; Development; Elderly; Environment; Epitopes; Equipment; Fc Receptor; Flow Cytometry; Future; Glycoproteins; Glycosaminoglycans; Goals; Grant; Health Priorities; Hospitalization; Human; Immune system; Infant; Infection; Injection of therapeutic agent; Institutes; Integration Host Factors; Knowledge; Ligands; Lower Respiratory Tract Infection; Mass Spectrum Analysis; Mediating; Membrane Fusion; Mentors; Molecular; Molecular Conformation; Monoclonal Antibodies; Morbidity - disease rate; Palivizumab; Physicians; Population; Preventive; Process; Proteins; Reagent; Recruitment Activity; Research; Resources; Respiratory Syncytial Virus Infections; Respiratory Tract Infections; Respiratory syncytial virus; Roentgen Rays; Role; Scientist; Shapes; Structure; Testing; Therapeutic; Time; Translating; Vaccine Antigen; Vaccines; Variant; Viral; Viral Fusion-GP; Virus Diseases; base; biophysical properties; dosage; global health; high risk infant; improved; inhibiting antibody; inhibitor/antagonist; insight; mortality; neutralizing antibody; neutralizing monoclonal antibodies; novel; nucleolin; prevent; prophylactic; receptor; receptor binding; research clinical testing; research study; screening; small molecule; tool; vaccine development

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infections that result in substantial morbidity and mortality in infants and the elderly. RSV entry into the host cell is facilitated by a fusion (F) glycoprotein that in its active form adopts a metastable prefusion conformation. After attachment of F to host-cell factors, it is hypothesized that one or more of these factors trigger the conformational rearrangement that results in fusion of the viral and cellular membranes. Since F is essential for RSV infection, humans elicit neutralizing antibodies that target it, with the most potent recognizing the prefusion conformation. Thus, this conformation of F is considered to be the ideal vaccine antigen, and antibodies and small molecules that disrupt its structure and function are rapidly being pursued. Development of effective therapeutics will be greatly enhanced by a molecular understanding of how the F glycoprotein interacts with host-cell factors that promote entry, and how neutralizing antibodies are able to inhibit one or more steps in the entry process. Through a collaborative effort we have determined X-ray crystal structures of the pre- and postfusion conformations of RSV F, stabilized a soluble form of the prefusion conformation, and identified a new class of potent neutralizing antibodies. This proposal builds upon these results by leveraging our prefusion-stabilized F glycoprotein and panel of antibodies to test the hypothesis that host-cell factors trigger RSV F rearrangement and that the most potent neutralizing antibodies target receptor binding sites and block conformational changes. In Aim 1, we propose to characterize specific interactions between prefusion F and host-cell factors, and determine which factors trigger F. In Aim 2, we plan to structurally determine the epitopes of F-directed neutralizing antibodies, and determine which steps in the viral entry process these antibodies inhibit. These studies will be greatly facilitated by the interactions and resources provided by the iTarget COBRE. Our mass spectrometry-based identification of RSV F receptors will benefit from interactions with Dr. Kettenbach, and our development of structure-based antivirals and their clinical testing will be shaped through interactions with Drs. Grigoryan and Ashare, a physician scientist. The Molecular Tools Core will provide support for the expression and purification of F glycoproteins, receptors, and antibodies. The functional and biophysical characterization of these proteins and their interactions will be facilitated by the Visualizing Molecular Interactions Core through access to SPR, ITC, FP, X-ray and flow cytometry equipment. The proposed project will advance our molecular understanding of the activation and inhibition of viral fusion glycoproteins by discovering and defining interactions of host factors with RSV F. Through involvement with the iTarget COBRE, this knowledge will be translated into the screening and development of RSV entry inhibitors, vaccine antigens and prophylactic antibodies. The COBRE will also provide an intellectual environment and mentoring support that will allow my research, and my lab, to expand and flourish.

呼吸道合胞病毒(RSV)引起急性下呼吸道感染，导致 婴儿和老年人的发病率和死亡率很高。RSV进入宿主细胞是通过一种 融合(F)糖蛋白，其活性形式采用亚稳定的融合前构象。在附着F之后 对于宿主细胞因子，假设这些因子中的一个或多个触发构象 导致病毒和细胞膜融合的重排。由于F对RSV感染是必不可少的， 人类会产生针对它的中和抗体，其中最有效的是识别融合前的构象。 因此，F的这种构象被认为是理想的疫苗抗原、抗体和小分子 破坏其结构和功能的行为正在迅速受到追查。有效疗法的开发将是 通过对F糖蛋白如何与宿主细胞因子相互作用的分子理解大大增强了这一点 促进进入，以及中和抗体如何能够抑制进入过程中的一个或多个步骤。 通过共同努力，我们已经确定了聚变前后的X射线晶体结构 RSVF的构象，稳定了一种可溶性的预融合构象，并鉴定了一类新的 强大的中和抗体。这项建议是在这些结果的基础上，通过利用我们的灌流稳定的F 用糖蛋白和一组抗体来检验宿主细胞因子触发RSV F重排的假设 最有效的中和抗体靶向受体结合部位并阻断构象 改变。在目标1中，我们建议表征预融合F和宿主细胞因子之间的特定相互作用， 并确定哪些因素会触发F。在目标2中，我们计划从结构上确定F导向的表位 中和抗体，并确定这些抗体抑制病毒进入过程中的哪些步骤。 ITarget提供的互动和资源将极大地促进这些研究 科布雷。我们基于质谱学的RSV F受体鉴定将受益于与Dr。 Kettenbach和我们基于结构的抗病毒药物的开发及其临床试验将通过 与内科科学家格里戈里安博士和沙雷博士的互动。分子工具核心将提供 支持F糖蛋白、受体和抗体的表达和纯化。功能和 这些蛋白质的生物物理特征及其相互作用将通过可视化得到促进 通过SPR、ITC、FP、X射线和流式细胞仪设备获得分子相互作用核心。 这项拟议的项目将促进我们对病毒激活和抑制的分子理解 通过发现和确定宿主因子与RSV F的相互作用来融合糖蛋白 有了iTarget Cobre，这些知识将转化为RSV进入的筛查和开发 抑制剂、疫苗抗原和预防性抗体。科布雷还将提供一名知识分子 环境和指导支持，这将使我的研究和我的实验室扩大和蓬勃发展。