Project 4: Molecular Mechanisms of RSV F Activation and Inhibition

呼吸道合胞病毒（RSV）引起急性下呼吸道感染， 婴儿和老年人的发病率和死亡率很高。RSV进入宿主细胞是由一种 融合（F）糖蛋白，其活性形式采用亚稳定的融合前构象。在连接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变化。 重组导致病毒和细胞膜的融合。由于F是RSV感染所必需的， 人类会引发针对它的中和抗体，其中最有效的是识别融合前构象。 因此，F的这种构象被认为是理想的疫苗抗原，以及抗体和小分子。 破坏其结构和功能的新技术正在迅速地加以研究。开发有效的治疗方法将是 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， 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。 通过合作，我们已经确定了融合前和融合后的X射线晶体结构。 RSV F的构象，稳定了融合前构象的可溶性形式，并鉴定了一类新的 强效中和抗体该提案通过利用我们的预融合稳定的F 糖蛋白和一组抗体，以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位点并阻断构象 变化在目的1中，我们提出了融合前F和宿主细胞因子之间的特异性相互作用的特征， 并确定哪些因素触发F。在目标2中，我们计划从结构上确定F-定向的表位。 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 眼镜蛇我们基于质谱的RSV F受体鉴定将受益于与Dr. 凯滕巴赫，我们的结构为基础的抗病毒药物的发展和他们的临床试验将通过以下方式形成 Grigoryan博士和Ashare博士的互动，一位医生科学家。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。的功能和 这些蛋白质及其相互作用的生物物理特性将通过可视化来促进。 分子相互作用核心通过访问SPR，ITC，FP，X射线和流式细胞仪设备。 该项目将促进我们对病毒激活和抑制的分子理解， 通过发现和定义宿主因子与RSV F的相互作用来研究融合糖蛋白。通过参与 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。眼镜蛇还将提供 环境和指导支持，这将使我的研究，我的实验室，扩大和蓬勃发展。

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.