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: 2066503
• 负责人: Mark Jay Yeager
• 金额: $ 21.88万
• 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/2066503
• 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.

呼肠孤病毒科包括无包膜的球形病毒颗粒，700-1100 直径有角，通常由同心的蛋白质壳形成 具有T=13/二十面体对称性，其对称性为10-12（1-4 kb） dsRNA基因组的片段。 我们的结构研究集中在两个方面 呼肠孤病毒科成员、轮状病毒和呼肠孤病毒。 轮状 感染导致严重的婴儿肠胃炎， 这是发展中国家婴儿死亡的主要原因。 尽管未 作为一种人类病原体，与呼肠孤病毒密切相关的呼肠孤病毒一直是 研究病毒性传染病发病机理的重要模型系统 疾病 轮状病毒和呼肠孤病毒的感染需要附着 表面血凝素蛋白（轮状病毒中的VP 4和呼肠孤病毒中的sigma 1） 肠道细胞表面的受体。 对于轮状病毒，感染 由胰蛋白酶切割VP 4介导，产生VP 5 * 和VP 8 *。 为 呼肠孤病毒、细胞和组织嗜性由σ 1蛋白赋予 这对于血清型1（Lang）和3（Dearing）是不同的。 我们最近 使用冷冻电子显微镜和二十面体图像重建， 推导出恒河猴轮状病毒的三维结构， 其保留了蛋白质和核酸的天然构象状态 酸 密度图中丰富的细节证明了这一点的力量 揭示复杂大分子结构的技术 结构. 我们的目标是确定超分子结构 以及设计几种不同的轮状病毒和呼肠孤病毒颗粒 当纯化和结晶VP 4时： 1. 轮状病毒结构分析 a. 天然与无刺突（即，缺乏血凝素VP 4） 轮状 B. 比较SA 11株和恒河猴轮状病毒，以检查 VP4 C. 比较天然轮状病毒和胰蛋白酶切割轮状病毒的结构 D. 检查内衣壳蛋白VP 6的晶体管和片层 e. 使用针对抗体的Fab片段进行免疫标记。 VP 4、VP 5 * 和VP 8 * 的胰蛋白酶裂解产物 F. 全面检查（即，含有dsRNA基因组）和空轮状病毒 核 2. 呼肠孤病毒结构分析 a. 比较天然呼肠孤病毒、胰蛋白酶切割的呼肠孤病毒的结构 （中间亚病毒颗粒[ISVP]）和呼肠孤病毒核心 B. 比较血清型1（Lang）和3（Dearing）呼肠孤病毒的结构 3. 纯化和结晶轮状病毒血凝素VP 4 我们的分析提供的结构信息将是基本的 为了全面了解病毒发病机制， 为合理设计治疗方案提供重要线索 战略布局