SUPRAMOLECULAR STRUCTURE AND DESIGN OF THE REOVIRIDAE

呼肠孤病毒科包括无包膜球形病毒颗粒700-1100 直径约1埃，通常由同心蛋白质壳形成 具有T=13/二十面体对称性，包括10-12(1-4kb) DsRNA基因组的片段。我们的结构研究主要集中在两个方面 呼肠孤病毒科、轮状病毒和呼肠孤病毒家族的成员。轮形病毒 感染会导致严重的婴儿胃肠炎，是主要的 导致发展中国家婴儿死亡的原因。虽然不是 作为一种人类病原体，密切相关的呼肠孤病毒一直是 研究病毒感染发病机制的重要模型系统 疾病。被轮状病毒和呼肠孤病毒感染需要附着 表面血凝素蛋白(轮状病毒的VP4和呼肠孤病毒的Sigma1) 到肠道细胞表面的受体。对于轮状病毒，感染 通过胰酶裂解VP4，产生VP5*和VP8*。为 呼肠孤病毒、细胞和组织的嗜性是由sigma1蛋白赋予的。 在1型(朗氏)和3型(德尔林)中，这是不同的。我们最近 使用冷冻电子显微镜和二十面体图像重建 条件下恒河猴轮状病毒三维结构的推导 它保持了蛋白质和核酸的天然构象状态 酸。密度贴图中丰富的细节展示了这一点的力量 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 并设计了几种不同的轮状病毒和呼肠孤病毒颗粒 作为纯化和结晶的VP4： 1.轮状病毒结构分析 A.天然与无刺(即缺乏血凝素VP4) 轮形病毒 B.比较SA11株和恒河猴轮状病毒，以检查在 VP4 C.比较天然和胰酶裂解轮状病毒的结构 D.检查内衣壳蛋白VP6的结晶管和晶片 E.使用针对Fab片段的免疫标记 VP4、VP5*和VP8*的胰酶切割产物 F.检查完整轮状病毒(即包含dsRNA基因组)和空轮状病毒 磁芯 2.呼肠孤病毒结构分析 A.比较天然呼肠孤病毒和胰酶裂解呼肠孤病毒的结构 (中间亚病毒颗粒[ISVP])和呼肠孤病毒核心 1型呼肠孤病毒(Lang)和3型呼肠孤病毒(Dring)的结构比较 3.轮状病毒血凝素VP4的纯化和结晶 我们的分析提供的结构信息将是基本的 以全面了解病毒的致病机制，并可能 为合理设计治疗性药物提供重要线索 战略。

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.