SUPRAMOLECULAR STRUCTURE AND DESIGN OF THE REOVIRIDAE

呼肠孤病毒科的超分子结构和设计

• 批准号: 2066502
• 负责人: Mark Jay Yeager
• 金额: $ 19.23万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-12-01 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2066502
• 关键词: Orthoreovirus; Rotavirus; capsid; chemical cleavage; computer simulation; conformation; cryopreservation; cryoscopy; crystallization; epitope mapping; genetic strain; image processing; immunoaffinity chromatography; microorganism hemagglutinin; neutralizing antibody; nucleocapsid; protein purification; protein structure function; proteolysis; virion; virus morphology; virus protein

The Reoviridae include non-enveloped spherical virus particles, 700-1100 Angstroms in diameter, generally formed by concentric protein shells having T=13/icosahedral symmetry, which encapsidate 10-12 (1-4 kb) segments of a dsRNA genome. Our structural studies are focussed on two members of the family of Reoviridae, rotavirus and reovirus. Rotavirus infection results in severe infantile gastroenteritis and is the major cause of human infant mortality in developing countries. Although not a human pathogen, the closely related reoviruses have served as an important model system for studying the pathogenesis of viral infectious diseases. Infection by rotavirus and reovirus requires attachment of the surface hemagglutinin proteins (VP4 in rotavirus and sigma1 in reovirus) to surface receptors on cells lining the gut. For rotavirus, infection is mediated by trypsin cleavage of VP4, generating VP5* and VP8*. For reovirus, cell and tissue tropism are conferred by the sigma1 protein which is different for serotypes 1 (Lang) and 3 (Dearing). We recently used cryo-electron microscopy and icosahedral image reconstruction to derive the 3-dimensional structure of rhesus rotavirus under conditions which preserve the native conformational state of the protein and nucleic acid. The rich detail in the density maps demonstrates the power of this technique to reveal the architecture of complex macromolecular structures. Our objectives are to determine the supramolecular structure and design of several different rotavirus and reovirus particles as well as purify and crystallize VP4: 1. Rotavirus structure analysis a. Native versus spikeless (i.e., lacking the hemagglutinin VP4) rotavirus b. Compare strain SA11 and rhesus rotavirus to examine differences in VP4 c. Compare the structures of native and trypsin-cleaved rotavirus d. Examine crystalline tubes and sheets of the inner capsid protein, VP6 e. Perform immunolabeling using Fab fragments directed against the trypsin cleavage products of VP4, VP5* and VP8* f. Examine full (i.e., containing the dsRNA genome) and empty rotavirus cores 2. Reovirus structure analysis a. Compare the structures of native reovirus, trypsin-cleaved reovirus (intermediate subviral particles [ISVPs]) and reovirus cores b. Compare the structures of serotypes 1 (Lang) and 3 (Dearing) reovirus 3. Purify and crystallize VP4, the rotavirus hemagglutinin The structural information provided by our analyses will be fundamental for a complete understanding of mechanisms of viral pathogenesis and may provide important clues for the rational design of therapeutic strategies.

旋旋菌科包括非发育的球形病毒颗粒，700-1100 直径的埃绳，通常由同心蛋白壳形成 具有t = 13/二十面体对称性，封装10-12（1-4 kb） DSRNA基因组的片段。 我们的结构研究集中在两个 Reoviridae，轮状病毒和异病毒家族的成员。 轮状病毒 感染导致严重的婴儿胃肠炎，是主要的 发展中国家人类婴儿死亡率的原因。 虽然不是 人类病原体，密切相关的肠道病毒已成为 研究病毒感染的发病机理的重要模型系统 疾病。 轮状病毒和依默病毒的感染需要附着 表面血凝素蛋白（轮状病毒中的VP4和eo依病毒中的Sigma1） 以表面受体在肠道内的细胞上。 对于轮状病毒，感染 由VP4的胰蛋白酶切割介导，生成VP5*和VP8*。 为了 sigma1蛋白赋予葡萄病毒，细胞和组织端主 对于血清型1（lang）和3（亲爱的）而言，这是不同的。 我们最近 使用的冷冻电子显微镜和二十面体图像重建 在条件下得出轮状病毒的三维结构 保留蛋白质和核糖的天然构象状态 酸。 密度图中的丰富细节证明了这一点的力量 揭示复杂大分子的结构的技术 结构。 我们的目标是确定超分子结构 以及几个不同的轮状病毒和依翔病毒颗粒的设计 如纯化和结晶VP4： 1。轮状病毒结构分析 一个。 天然与尖刺（即缺乏hemagglutinin vp4） 轮状病毒 b。 比较菌株SA11和轮状病毒的菌株以检查 VP4 c。 比较天然和胰蛋白酶切割的轮状病毒的结构 d。 检查内膜蛋白的结晶管和板VP6 e。 使用针对的Fab片段进行免疫标签 VP4，VP5*和VP8*的胰蛋白酶切割产品 f。 检查完整（即包含DSRNA基因组）和空轮状病毒 内核 2。旋孢病毒结构分析 一个。 比较原生蠕虫病毒，胰蛋白酶裂解的葡萄病毒的结构 （中级次病颗粒[ISVPS]）和静脉病毒核心 b。 比较血清型1（lang）和3（亲爱的）异病毒的结构 3。纯化和结晶VP4，轮状病毒血凝素 我们分析提供的结构信息将是基本的 为了完全了解病毒发病机理的机制，可能 为理性设计提供重要的线索 策略。