Investigation of the biophysical mechanisms and membrane determinants of respirovirus binding and fusion using artificial lipid membranes and isolated physiological membranes as targets

项目总结/摘要 该项目的主要目标是：（A）研究生物物理机制的初始步骤， 呼吸道病毒感染（与宿主细胞结合并随后进行膜融合），和B）开发/验证生物物理 用于更广泛地研究病毒结合/融合的方法。呼吸道病毒是副粘病毒家族的成员，并且 是呼吸道感染的主要原因，特别是在儿童中。尽管呼吸道病毒对健康的影响 由于负担沉重，迄今为止还没有获得许可的呼吸道病毒特异性抗病毒药物或疫苗。更深入地了解 呼吸道病毒感染机制对于开发治疗方法将是重要的。研究呼吸道病毒结合， 融合，该项目将使用鼠呼吸道病毒（a.k.a.仙台病毒），其已被用作呼吸道病毒的模型， 一段时间除了其作为模型呼吸道病毒的效用之外，仙台病毒本身也引起了相当大的兴趣-其广泛的 在人类细胞中的组织嗜性和在人类中缺乏致病性使其成为有吸引力的临床和实验室载体 用于基因治疗和疫苗开发。因此，本研究的目的不仅是扩大对机械的理解 呼吸道病毒的结合和融合，而且也是这个有用的实验室工具。 为了研究呼吸道病毒结合和融合的生物物理机制， 采用，观察单个病毒颗粒和宿主细胞膜模拟物（称为模型脂质）之间的相互作用 膜。模型脂质膜是由一些脂质成分形成的脂质双层，允许实验者 研究关键的分子相互作用。在目标1中，这种方法将用于研究 病毒与宿主细胞膜上的受体结合，这种结合如何触发病毒启动膜 以及胆固醇在这些过程中所起的作用。 AIM 2将开发和使用分析化学方法来研究模型的化学成分 膜本身。模型脂膜不仅在本项目中用作宿主细胞膜模拟物， 也在许多领域用作细胞膜模拟物，包括药物递送、药物配制和基础科学研究。 它们可以让实验者精确控制膜的组成，但很少有研究人员能够检查 由其制备方法产生的组合物，特别是在单一病毒研究中。因此，该项目将 确定最佳实践，以最大限度地减少变异性，并在单一病毒研究和其他领域产生可靠的结果。 最后，本项目还旨在开发新的模型膜平台， 红细胞（Aim 3）。这些平台将被用来调查其他生物物理问题的影响， 膜环境对呼吸道病毒结合和融合的影响，并将能够精确控制和测量单个 病毒在天然膜环境中的作用。它们也将更广泛地用于研究病毒结合， 其他病毒的融合。

PROJECT SUMMARY/ABSTRACT The broad objectives of this project are A) to investigate the biophysical mechanisms of the initial steps of respirovirus infection (binding to the host cell and subsequent membrane fusion), and B) to develop/validate biophysical methods used to study viral binding/fusion more broadly. Respiroviruses are members of the paramyxovirus family, and are a leading cause of respiratory infection, especially in children. Although respiroviruses present a significant health burden, there are no licensed respirovirus-specific antivirals or vaccines to date. Deeper fundamental understanding of the respirovirus infection mechanisms will be important to develop therapeutic approaches. To study respirovirus binding and fusion, this project will use murine respirovirus (a.k.a. Sendai virus), which has been used as a model respirovirus for some time. Aside from its utility as a model respirovirus, Sendai virus itself is also of considerable interest – its wide tissue tropism in human cells and lack of pathogenicity in humans has made it an attractive clinical and laboratory vector for gene therapy and vaccine development. Therefore, this study aims not only to expand mechanistic understanding of respirovirus binding and fusion, but also of this useful laboratory tool. To investigate the biophysical mechanisms of respirovirus binding and fusion, single virus measurements will be employed, observing interactions between single virus particles and host cell membrane mimics called model lipid membranes. Model lipid membranes are lipid bilayers formed from a few lipid components, allowing the experimenter to investigate key molecular interactions. In Aim 1, this approach will be utilized to investigate the molecular mechanism of viral binding to its receptor in the host cell membrane, how that binding then triggers the virus to initiate membrane fusion, and the role that cholesterol plays in these processes. Aim 2 will develop and use analytical chemistry methods to study the chemical composition of the model membranes themselves. Model lipid membranes are not only used as host cell membrane mimics in this project, but are also used as cell membrane mimics in many fields, including in drug delivery, drug formulation, and basic science studies. They can afford the experimenter precise control over the membrane composition, but it is rare for researchers to examine the composition produced by their preparation method, especially in single virus studies. Therefore, this project will identify best practices to minimize variability and produce robust results both in single virus studies, and in other fields. Finally, this project also aims to develop new model membrane platforms, using membranes derived from human red blood cells (Aim 3). These platforms will be used to investigate additional biophysical questions about the influence of membrane environment on respirovirus binding and fusion, and will enable precise control and measurement of single viruses in the context of a native membrane environment. They will also be useful more broadly to study viral binding and fusion for other viruses as well.