MECHANISMS BY WHICH ROTAVIRUS INDUCES DIARRHEA

这项奖学金申请提出了研究，以了解致病机理 轮状病毒引起儿童严重腹泻的机制。 轮状病毒有一种独特的形态发生，其中粒子获得了 由新种植物萌发形成的瞬变膜被膜 亚病毒颗粒进入内质网(ER)。这个过程是 由一种名为NSP4的病毒非结构糖蛋白介导，它是 定位于感染细胞的内质网细胞膜。最近在这方面的研究 实验室已经证明，NSP4和一种合成肽NSP4 114.135 (对应于NSP4氨基酸114-135)，注射时 经腹膜内或十二指肠内注射，幼年小鼠腹泻， 老鼠。NSP4和NSP4 114.1350，当在其中表达或外源添加到 昆虫细胞，动员细胞内钙。我们推测NSP4可能是 引起沙门氏菌引起腹泻的毒力因子 NSP4的钙动员特性可能 构成病毒的一条信号转导通路 致病性。电生理方法和常规方法一样 在这一应用中提出了分子生物学技术来 阐明NSP4动员细胞内钙离子和 导致幼年动物腹泻。了解这些机制可能会 为解决轮状病毒的致病途径提供了关键。因为 对于这种轮状病毒非结构糖蛋白的独特性质，我们 有可能阐明病毒致病的新机制和新的 病毒引起钙动员的信号转导途径 蛋白质。在拟议的研究中，我将学习新技术，包括： Ussing小室、平面脂质双层、重组人的构建和应用 质粒或病毒、点特定突变和其他分子方法 有必要开展研究以回答以下基本问题 分子生物学。

This fellowship application proposes studies to understand the pathogenic mechanism by which rotavirus causes severe diarrhea in children. Rotavirnses have a unique morphogenesis in which particles obtain a transient membrane envelope that is formed by the budding of newly made subviral particles into the endoplasmic reticulum (ER). This process is mediated by a viral nonstructural glycoprotein called NSP4, which is localized to the ER membrane of infected cells. Recent studies in this laboratory have shown that NSP4 and a synthetic peptide NSP4 114.135 (corresponding to NSP4 amino acids 114-135), when injected intraperitoneally or intraduodenally, induced diarrhea in young mice and rats. NSP4 and NSP4 114.1350, when expressed in or added exogenously to insect cells, mobilize intracellular calcium. We speculate that NSP4 may be the virulence factor responsible for inducing the diarrhea caused by rotaviruses, and that the calcium mobilization property of NSP4 may underlie a signal transduction pathway which contributes to virus pathogenicity. Electrophysiological approaches as well as conventional molecular biological techniques are proposed in this application to delineate the mechanisms by which NSP4 mobilizes intracellular calcium and induces diarrhea in young animals. Understanding these mechanisms may provide the key to resolving the pathogenic pathway of rotaviruses. Because of the unique properties of this rotavirus nonstructural glycoprotein, we are likely to elucidate new mechanisms of viral pathogenesis and novel signal transduction pathways of calcium mobilization caused by viral proteins. In the proposed research, I will learn new techniques including: Ussing chamber, planar lipid bilayer, construction and use of recombinant plasmids or virus, point specific mutations, and other molecular approaches necessary to carry out research to answer fundamental questions in molecular biology.