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.

该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。 上皮细胞 细胞在体内组织成片，形成分隔的屏障 身体隔间。 这些上皮片的主要功能是移动 特定分子从一侧到另一侧。 为了实现这一目标，非常 不同的蛋白质群体必须在两个表面上表达。 毫不奇怪，病毒已经进化出使用此功能的策略，并且 许多动物病毒的感染和传播途径都反映了这一点 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 通过昆虫宿主传播，通过暴露于的表面感染并成熟 血。 上皮细胞系（例如 MDCK 细胞）在 体外，不同的蛋白质群位于顶端（顶部） 和基底外侧（底部）细胞表面。 大多数膜糖蛋白 感染这些细胞的包膜病毒专门针对一种 的表面。 已分离出几种突变 MDCK 细胞系， 当感染水泡性口炎病毒（VSV）时，会表现出缺陷 病毒包膜糖蛋白（G蛋白）适当成熟为 基底外侧表面。 相反，当这些突变体感染 流感病毒，其主要包膜糖蛋白（HA）正常成熟， 顶端表面。 其中四个突变体的初步表征 表明两个（克隆 32 和 39）允许 G 蛋白出现在顶端 （不正确的）表面，一个（克隆 43）在 内质网，一个（克隆 77）显示没有明显的 G-合成 蛋白质。 这些突变体的表型将被进一步表征。 分析 克隆 43 中生长的 G 蛋白的寡糖链将识别 其细胞积累的位点。l 克隆 32 和 32 号感染的单层细胞 39将通过3H-菊粉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。 已感染 克隆 77 的单层细胞将用多克隆抗血清进行免疫沉淀 以确定是否正在产生抗原性改变的 G 蛋白。 普遍性 将通过研究成熟度来检查突变表型的 细胞基底外侧蛋白和来自不相关病毒的基底外侧蛋白。 最终，细胞成分的鉴定和纯化 克隆 43 和 77 中存在缺陷的修复将使用无细胞来完成 重建系统作为测定。 连续纯化的提取物 野生型细胞将用于补充突变细胞裂解物以鉴定 对于“拯救”缺陷的重要因素。