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

项目摘要/摘要 本项目的主要目标是：A)研究以下最初步骤的生物物理机制 呼吸道病毒感染(与宿主细胞结合和随后的膜融合)，以及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。呼吸道合胞病毒是副粘病毒家族的成员， 是呼吸道感染的主要原因，特别是在儿童中。尽管呼吸道病毒表现出显著的健康状况 负担，到目前为止还没有获得许可的针对呼吸道病毒的抗病毒药物或疫苗。更深层次地理解 呼吸道病毒感染机制对开发治疗方法将是重要的。研究呼吸道病毒结合和 融合，这个项目将使用小鼠呼吸道病毒(又名。仙台病毒)，它已被用作呼吸道病毒的模型 过段时间吧。除了作为一种模式呼吸病毒的用途外，仙台病毒本身也引起了相当大的兴趣-它的广泛 在人体细胞中的组织嗜性和在人类中的无致病性使其成为一种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，本研究的目的不仅在于扩大对 呼吸道病毒的结合和融合，也是这一有用的实验室工具。 为了研究呼吸道病毒结合和融合的生物物理机制，将对单个病毒进行测量 用来观察单个病毒颗粒和宿主细胞膜模拟物之间的相互作用，称为模型脂质 膜。模型类脂膜是由几种类脂成分形成的类脂双层，允许实验者 研究关键的分子相互作用。在目标1中，这一方法将被用来研究 病毒与宿主细胞膜上的受体结合，然后这种结合如何触发病毒启动细胞膜 融合，以及胆固醇在这些过程中扮演的角色。 目标2将开发并使用分析化学方法来研究模型的化学成分 膜本身。在本项目中，模型类脂膜不仅用作宿主细胞膜的模拟物，而且还 在许多领域也用作细胞膜模拟物，包括药物输送、药物配方和基础科学研究。 它们可以为实验人员提供对膜成分的精确控制，但研究人员很少能检查 由它们的制备方法产生的组合物，尤指在单一病毒研究中。因此，这个项目将 确定最佳做法，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强有力的结果。 最后，该项目还旨在开发新型膜平台，使用来自人类的膜 红细胞(目标3)。这些平台将被用来调查有关影响的其他生物物理问题 膜环境对呼吸道病毒结合和融合的影响，并将使精确控制和测量单个 病毒在原生膜环境的背景下。它们还将在更广泛的范围内用于研究病毒结合和 其他病毒的融合也是如此。

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.