MDCK CELL MUTANTS DEFECTIVE IN GLYCOPROTEIN MATURATION

MDCK 细胞突变体糖蛋白成熟缺陷

• 批准号: 3436819
• 负责人: JEFFREY T MAYNE
• 金额: $ 10.02万
• 依托单位: VASSAR COLLEGE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3436819
• 关键词: Orthomyxoviridae; Vesiculovirus; biological transport; cell bank /registry; cell free system; clone cells; electron microscopy; epithelium; gene expression; glycoproteins; immunofluorescence technique; immunoprecipitation; membrane activity; membrane proteins; monoclonal antibody; mutant; protein biosynthesis; virus infection mechanism; virus protein

The major goal of this project is to investigate the cellular mechanisms involved in the polarized expression of cell surface proteins. Epithelial cells are organized into sheets in vivo, forming barriers that separate body compartments. A major function of these epithelial sheets is to move specific molecules from one side to the other. To accomplish this, very different populations of proteins must be expressed at the two surfaces. Not surprisingly, viruses have evolved strategies to use this feature, and the route of infection and spread of many animal viruses reflect this asymmetry. Viruses which spread through the respiratory tract mature from the epithelial cell surface exposed in the lungs, whereas viruses that are spread by insect hosts infect and mature through the surfaces exposed to the blood. Epithelial cell lines such as MDCK cells form polarized monolayers in vitro, with separate populations of proteins localized at the apical (top) and basolateral (bottom) cell surfaces. The membrane glycoproteins of most enveloped viruses that infect these cells are specifically targeted to one of the surfaces. Several mutant MDCK cell lines have been isolated which, when infected by vesicular stomatitis virus (VSV), exhibit defects in the proper maturation of the viral envelope glycoprotein (G-protein) to the basolateral surface. In contrast, when these mutants are infected with influenza virus, its major envelope glycoprotein (HA) matures properly to the apical surface. Preliminary characterization of four of these mutants indicate that two (clones 32 & 39) allow G-protein to appear at the apical (incorrect) surface, one (clone 43) accumulates G-protein in the endoplasmic reticulum, and one (clone 77) shows no apparent synthesis of G- protein. The phenotypes of these mutants will be further characterized. Analysis of the oligosaccharide chains of G-protein grown in clone 43 will identify the site of its cellular accumulation.l Infected monolayers of clones 32 and 39 will be tested by 3H-inulin diffusion and electron microscopic studies to determine whether the junctional complexes are intact. Infected monolayers of clone 77 will be immunoprecipitated with polyclonal antisera to determine if antigenic altered G-protein is being made. The generality of the mutant phenotype will be examined by studying the maturation of cellular basolateral proteins and those from unrelated viruses. Ultimately, identification and purification of of the cellular components that are defective in clones 43 and 77 will be accomplished using cell-free reconstitution systems as an assay. Sequentially purified extracts of wild-type cells will be used to supplement mutant cell lysates to identify the factors important for "rescue" of the defect.

这个项目的主要目标是研究细胞机制。 参与细胞表面蛋白的极化表达。上皮性 在体内，细胞被组织成片状，形成屏障，将 车身隔间。这些上皮膜的一个主要功能是移动 从一边到另一边的特定分子。要做到这一点，非常 不同的蛋白质群体必须在两个表面表达。 毫不奇怪，病毒已经进化出使用这一功能的策略，并且 许多动物病毒的感染和传播途径反映了这一点 不对称。通过呼吸道传播的病毒成熟于 暴露在肺部的上皮细胞表面，而病毒是 由昆虫宿主传播，通过暴露在 鲜血。 MDCK细胞等上皮细胞系在体内形成极化单层 体外培养，不同的蛋白质群体定位于顶端(顶部) 和基侧(底部)细胞表面。MOST的膜糖蛋白 感染这些细胞的包膜病毒是专门针对一种 表面上的。已分离出几个突变的MDCK细胞系， 当感染水疱性口炎病毒(VSV)时，表现出皮肤缺陷 病毒包膜糖蛋白(G蛋白)的适当成熟 基准面。相反，当这些突变体感染了 流感病毒，其主要包膜糖蛋白(HA)适当成熟以 顶面。其中4个突变体的初步鉴定 表明两个克隆(克隆32和39)允许G蛋白出现在顶端 (错误)表面，一个(克隆43)在细胞内积聚G蛋白 其中一个克隆(克隆体77)没有明显的G-合成。 蛋白。 这些突变体的表型将进一步确定。分析 在克隆43中生长的G蛋白的寡糖链将识别 它的细胞积累位置。l感染单层的克隆32和 39将通过~3H-菊糖扩散和电子显微镜研究进行测试 以确定连接复合体是否完整。受感染的 克隆77的单层将与多克隆抗血清进行免疫沉淀 以确定是否正在制造抗原性改变的G蛋白。概论 通过研究突变表型的成熟度来检测突变表型 细胞基侧蛋白和来自无关病毒的蛋白。 最终，对细胞成分的鉴定和纯化 克隆43和77中有缺陷的克隆将使用无细胞 重建系统作为一种分析。顺次提纯的丹参提取物 野生型细胞将被用来补充突变细胞裂解物以鉴定 对缺陷进行“挽救”的重要因素。