Characterization of the interaction of M1 and M2: Influenza A proteins critical to viral assembly

M1 和 M2 相互作用的表征：甲型流感蛋白对病毒组装至关重要

• 批准号: 9813240
• 负责人: KATHLEEN P HOWARD
• 金额: $ 36.18万
• 依托单位: SWARTHMORE COLLEGE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9813240
• 关键词: Acute; Address; Affect; Amino Acids; Antiviral Agents; Applications Grants; Binding; Binding Proteins; Biochemical; Biophysics; Calorimetry; Cell membrane; Cells; Circular Dichroism; Complex; Coupled; Data; Development; Electron Spin Resonance Spectroscopy; Electrons; Epidemic; Experimental Designs; Foundations; Future; Generations; Genetic; Healthcare; Influenza; Influenza A virus; Knowledge; Label; Lead; Length; Life Cycle Stages; Liposomes; Literature; M2 protein; Magnetic Resonance; Maps; Measures; Membrane; Membrane Lipids; Mentors; Molecular Biology; Molecular Conformation; Nuclear; Persons; Pharmaceutical Preparations; Play; Process; Proteins; Public Health; Publishing; Reagent; Recurrence; Resistance; Sampling; Site; Spin Labels; Structure; Students; Surface; Testing; Titrations; Viral; Viral Proteins; Virion; Virus; Virus Assembly; Virus Diseases; Virus Replication; Work; age group; anti-influenza drug; biophysical analysis; biophysical techniques; career development; design; experimental study; influenza M2; influenzavirus; insight; interdisciplinary collaboration; light scattering; overexpression; pandemic disease; pandemic influenza; protein reconstitution; recruit; undergraduate research

PROJECT SUMMARY/ABSTRACT Influenza A is an acute viral infection that spreads easily from person to person, can affect any age group and causes recurrent seasonal epidemics and global pandemics. Fundamental information on the influenza virus life cycle is still being discovered. In particular, there have been recent developments that provide new insights into how viruses assemble and then bud at the surface of infected cells. An atomic-level understanding of the viral assembly and budding process could lead to strategies to inhibit the replication of viruses and new tactics for inhibiting viral infectivity. Two of the proteins required for the efficient assembly of infectious virus particles are M1 and M2. Developing a detailed biophysical understanding of the interaction between M1 and M2 is the focus of this proposal. Due to the key role the M1-M2 interaction plays in viral assembly, it has been proposed that the disruption of this interaction could lead to a new class of antiviral drugs. The M1 protein is a 252-amino acid soluble protein and M2 protein is a 97-amino acid membrane-bound protein. Both localize to the plasma membrane of an infected cell prior to viral budding. A strength of the current state of knowledge is that published work on M1 and M2 provides the genetic, biochemical and molecular biology foundations necessary to make biophysical studies tractable. Our proposed work addresses a current weakness in the field – a lack of atomic level detail on the molecular interaction of these two key players in viral budding. A combination of different magnetic resonance approaches will be used to probe the interaction of M1 with M2. Using site-directed spin-label electron paramagnetic resonance (SDSL-EPR), we will measure the mobility of spin labels, accessibility of spin labels to collision with paramagnetic reagents and distance dependent spin couplings. The PI’s lab can already efficiently biochemically produce and spectroscopically characterize spin-labeled full-length M2 protein reconstituted into liposomes. We have already biochemically overexpressed wild-type M1 and we describe our proposed strategy for spin-labeling M1. We propose to characterize the interaction between M1 and M2 by measuring distances between spin- labeled M1 and spin-labeled M2. We also plan to introduce site-specific 19F labels and measure distances using strategies that rely on the presence of both nuclear and electron spins. Additional biophysical techniques (differential light scattering, circular dichroism and isothermal titration calorimetry) will also be used to characterize the M1-M2 complex. Undergraduate research students supported by this grant proposal will explore an issue of critical public health importance using cutting edge biophysical techniques, participate in established interdisciplinary collaborations, be co-authors on published work and be mentored by experts committed to their long-term career development.

项目摘要/摘要 流感是一种急性病毒感染，可轻松地从人到人传播，可能会影响任何年龄 小组并导致经常性的季节性发作和全球大流行。有关 流感病毒生命周期仍在发现。特别是，最近有一些发展 提供有关病毒如何组装然后在感染细胞表面芽的新见解。原子水平 了解病毒组装和发芽过程可能会导致抑制复制的策略 病毒和抑制病毒感染的新策略。 有效组装感染性病毒颗粒所需的两个蛋白质为M1和M2。 对M1和M2之间的相互作用进行详细的生物物理理解是其中的重点 提议。由于M1-M2相互作用在病毒组装中的关键作用，因此已经提出了 这种相互作用的破坏可能导致新的抗病毒药。 M1蛋白是252-氨基酸 可溶性蛋白和M2蛋白是一种97-氨基酸膜结合的蛋白。两者都本地化到等离子体 在病毒萌芽之前，感染细胞的膜。当前知识状态的优势是 在M1和M2上发表的工作提供了必要的遗传，生化和分子生物学基础 使生物物理研究可进行。我们提出的工作解决了该领域的当前弱点 - 缺乏 关于这两个关键参与者在病毒萌芽中的分子相互作用的原子级别的细节。 不同的磁共振方法的组合将用于探测M1的相互作用 与M2。使用位置定向的自旋标签电子顺磁共振（SDSL-EPR），我们将测量 自旋标签的活动性，自旋标签与顺磁试剂碰撞和距离的可及性 依赖性自旋耦合。 PI的实验室已经可以有效地在生物化学上产生并在光谱上产生 表征了自旋标记的全长M2蛋白，重构为脂质体。我们已经在生化上 过表达的野生型M1，我们描述了我们提出的自旋标记M1的策略。 我们建议通过测量自旋之间的距离来表征M1和M2之间的相互作用 标记的M1和自旋标记的M2。我们还计划使用特定于现场的19F标签和测量距离使用 依靠核和电子自旋的存在的策略。其他生物物理技术 （差分光散射，圆形二色性和等温滴定量热法）也将用于 表征M1-M2复合物。 该赠款建议支持的本科研究学生将探讨一个关键的问题 使用尖端生物物理技术的公共卫生重要性，参与已建立的跨学科 合作，成为出版工作的合着者，并由致力于长期的专家至关重要 职业发展。