RESPIRATORY VIRUS ION CHANNELS

呼吸道病毒离子通道

• 批准号: 2066831
• 负责人: LAWRENCE H PINTO
• 金额: $ 14.64万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2066831
• 关键词: Orthomyxoviridae; Xenopus; Xenopus oocyte; acidity /alkalinity; alternatives to animals in research; amantadine; calcium; conformation; drug resistance; glycoproteins; glycosylation; immunocytochemistry; immunoprecipitation; ion transport; lipid bilayer membrane; membrane channels; membrane potentials; membrane reconstitution /synthesis; mutant; posttranslational modifications; protein sequence; protein structure function; radioimmunoassay; site directed mutagenesis; tissue /cell culture; transfection; virus infection mechanism; virus protein; virus replication; voltage /patch clamp

Influenza continues to be a regular epidemic, and occasionally pandemic, disease which ranks among the leading causes of morbidity and mortality throughout the world, particularly in the elderly and in individuals with heart and lung disease. Influenza viruses encode two small integral membrane proteins of similar and unusual structure, the M2 protein of influenza A virus and the NB glycoprotein of influenza B virus. The proposed research is based upon our demonstration that the M2 protein forms an ion channel and our finding that ion channel activity is associated with the NB protein. (1) We propose to study the structure- function relationship of the M2 protein by making measurements of ion channel activity (including specific activity) of site-directed mutants modified to render them insensitive to the antiviral drug amantadine or lacking in certain post-translational modifications. We also propose to create an active ion channel of the minimal size and to reconstitute ion channel activity in lipid bilayers. (2) The role of the ion channel activity of the M2 protein in the life-cycle of the influenza A virus will be characterized by measuring the pH-activation of the ion channel in mammalian cells that express the protein, comparing the pH-activation of the M2 proteins of various strains of influenza A virus having hemagglutinin (HA) molecules which undergo conformational changes to the acid state at different values of pH and by comparing the ion channel properties of M2 proteins derived from "high yield" strains of influenza A virus. (3) The structure-function relationship of the NB-associated ion channel activity will be studied. We will compare the properties of the ion channel activity associated with the wild-type and various mutant forms of the NB protein to determine if the NB protein is itself an ion channel. The roles of carbohydrate addition, Ca2+ regulation and the polar residues in the transmembrane domain in the function of the NB protein will be investigated using analysis of site-specific mutants. The specific activity of the NB-associated ion channel activity will be measured, the minimal protein needed for channel activity will be determined, and the channel activity reconstituted in lipid bilayers. These studies will provide essential information about the role of the M2 and NB proteins that may help target them for drug action and will provide basic information upon which future structural (e.g., NMR and crystallographic) studies of these minimal channels can be based.

流感仍然是一种常规流行病，偶尔也会大流行， 疾病是发病和死亡的主要原因之一 在世界各地，特别是在老年人和有 心脏和肺部疾病。 流感病毒编码两个小的整数 膜蛋白的相似和不寻常的结构，M2蛋白的 流感A病毒和流感B病毒的NB糖蛋白。 的 我们提出的研究是基于我们证明M2蛋白 形成了一个离子通道，我们发现离子通道的活性 与NB蛋白有关。 (1)我们建议研究结构- M2蛋白的功能关系 定点突变体的通道活性（包括比活性） 修饰使其对抗病毒药物金刚烷胺不敏感，或 缺乏某些翻译后修饰。 我们亦建议 产生最小尺寸的活性离子通道， 脂质双层中的通道活性。 (2)离子通道的作用 M2蛋白在甲型流感病毒生命周期中的活性 将通过测量离子通道的pH活化来表征 在表达该蛋白的哺乳动物细胞中， 不同甲型流感病毒株的M2蛋白中， 血凝素（HA）分子，其经历构象变化， 不同pH值下的酸状态并通过比较离子通道 来源于“高产”流感病毒株的M2蛋白的性质 一种病毒 (3)NB相关蛋白的结构与功能关系 将研究离子通道活性。 我们将比较 与野生型和各种突变体相关的离子通道活性 以确定NB蛋白本身是否是离子 频道 碳水化合物的添加、Ca ~（2+）的调节和 跨膜结构域中的极性残基在NB的功能中 将使用位点特异性突变体的分析来研究蛋白质。 NB相关离子通道活性的比活性为 测量，通道活性所需的最小蛋白质将是 测定，并在脂质双层中重建通道活性。 这些研究将提供关于 M2和NB蛋白可能有助于靶向药物作用， 提供未来结构（例如，NMR和 晶体学）研究这些最小通道。