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

以人工脂质膜和分离的生理膜为靶标，研究呼吸道病毒结合和融合的生物物理机制和膜决定因素

• 批准号: 10513664
• 负责人: Robert J. Rawle
• 金额: $ 39.29万
• 依托单位: WILLIAMS COLLEGE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10513664
• 关键词: Address; Analytical Chemistry; Animals; Antiviral Agents; Basic Science; Behavior; Binding; Biological Assay; Biophysical Process; Biophysics; Cell Culture Techniques; Cell membrane; Cells; Chemicals; Child; Cholesterol; Clinical; Coupled; Data; Drug Delivery Systems; Drug Formulations; Environment; Erythrocytes; Event; Family; Fluorescence; Gene Transduction Agent; Health; Human; Infection; Investigation; Knowledge; Laboratories; Lipid Bilayers; Lipids; Liposomes; Measurement; Mediating; Membrane; Membrane Fusion; Membrane Lipids; Methods; Modeling; Molecular; Molecular Conformation; Mus; Para-Influenza Virus Type 1; Paramyxovirus; Pathogenicity; Physiological; Play; Preparation; Process; Proteins; Research Personnel; Respiratory Disease; Respiratory Tract Infections; Respirovirus; Respirovirus Infections; Role; Sendai virus; Techniques; Therapeutic; Time; Vaccines; Viral; Viral Fusion Proteins; Virion; Virus; Virus Diseases; biophysical analysis; biophysical techniques; experimental study; human pathogen; insight; interest; kinetic model; member; membrane model; parainfluenza virus; receptor; respiratory; stoichiometry; therapy development; tissue tropism; tool; undergraduate research; vaccine development

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.

项目摘要/摘要 该项目的广泛目标是a）研究的生物物理机制 呼吸病毒感染（与宿主细胞和随后的膜融合结合），b）发展/验证生物物理 用于更广泛地研究病毒结合/融合的方法。呼吸病毒是Paramyxovirus家族的成员， 虽然呼吸病毒表现出色 负担，迄今为止，没有许可的呼吸病毒特异性抗病毒药或疫苗。对 呼吸病毒感染机制对于开发治疗方法很重要。研究呼吸病毒结合和 融合，该项目将使用鼠呼吸病毒（又称仙台病毒），该病毒已被用作模型呼吸病毒 一段时间。除了其作为模型呼吸病毒的实用性外，仙台病毒本身也具有考虑 - 广泛 人类细胞中的组织向往往和人类缺乏致病性使其成为有吸引力的临床和实验室载体 用于基因疗法和疫苗发育。因此，这项研究不仅旨在扩大对 呼吸病毒结合和融合，也是这种有用的实验室工具。 为了研究呼吸病毒结合和融合的生物物理机制，单个病毒测量将是 使用的，观察单个病毒颗粒与宿主细胞膜模拟物之间的相互作用称为模型脂质 膜。脂质膜模型是由几个脂质成分形成的脂质双层，使专家能够 研究关键分子相互作用。在AIM 1中，该方法将用于研究 病毒结合与宿主细胞膜中的受体结合，该结合如何触发病毒启动膜 融合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组成 膜本身。模型脂质膜不仅用作宿主细胞膜模仿该项目，而且是 还用作许多领域的细胞膜模拟物，包括药物输送，药物配方和基础科学研究。 他们可以负担对膜成分的专家精确控制，但是研究人员很少检查 通过其制备方法产生的组成，尤其是在单个病毒研究中。因此，这个项目将 确定最佳实践，以最大程度地减少变异性并在单个病毒研究和其他领域产生强大的结果。 最后，该项目还旨在使用人类的机制开发新的模型膜平台 红细胞（目标3）。这些平台将用于调查有关影响的其他生物物理问题 在呼吸病毒结合和融合上的膜环境，并可以精确控制和测量 在本地膜环境的背景下的病毒。它们还将对研究病毒结合和 其他病毒也融合。

项目成果

Viral Size Modulates Sendai Virus Binding to Cholesterol-Stabilized Receptor Nanoclusters.

• DOI: 10.1021/acs.jpcb.2c03830
• 发表时间: 2022-09-15
• 期刊: JOURNAL OF PHYSICAL CHEMISTRY B
• 影响因子: 3.3
• 作者: Lam, Amy;Yuan, Daniel S.;Ahmed, Samir H.;Rawle, Robert J.
• 通讯作者: Rawle, Robert J.

Choice of buffer in mobile phase can substantially alter peak areas in quantification of lipids by HPLC-ELSD.

• DOI: 10.1016/j.jchromb.2022.123417
• 发表时间: 2022-10-15
• 期刊: JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
• 影响因子: 3
• 作者: Graceffa, Oliva;Kim, Eunice;Broweleit, Rachel;Rawle, Robert J.
• 通讯作者: Rawle, Robert J.