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.

本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 细胞在体内被组织成片状，形成屏障， 身体隔间 这些上皮片层的主要功能是移动 从一侧到另一侧。 为了实现这一点，非常 必须在两个表面上表达不同的蛋白质群体。 毫不奇怪，病毒已经进化出利用这一特征的策略， 许多动物病毒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从 暴露在肺中的上皮细胞表面，而暴露在肺中的 由昆虫宿主传播，感染并通过暴露于 血 上皮细胞系如MDCK细胞在细胞内形成极化单层， 体外，位于顶端的蛋白质的单独群体（顶部） 和基底外侧（底部）细胞表面。 大多数人的膜糖蛋白 感染这些细胞的包膜病毒特异性地针对一种 的表面。 已经分离出几种突变MDCK细胞系， 当被水泡性口炎病毒（VSV）感染时， 病毒包膜糖蛋白（G蛋白）的适当成熟， 基底外侧表面。 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， 顶面 这些突变体中的四个的初步表征 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）积累G蛋白在 内质网，其中一个（克隆77）没有明显的G-合成 蛋白 这些突变体的表型将进一步表征。 分析 克隆43中生长的G蛋白的寡糖链将鉴定 l克隆32和34的感染单层细胞， 39将通过3 H-菊粉扩散和电子显微镜研究进行检测 以确定连接复合体是否完整。 感染 克隆77的单层将用多克隆抗血清免疫沉淀 以确定是否有抗原性改变的G蛋白被制造出来 一般性 突变表型的研究将通过研究 细胞基底外侧蛋白和来自无关病毒的蛋白。 最终，鉴定和纯化细胞组分 克隆43和77中的缺陷将使用无细胞 作为测定的重构系统。 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， 对缺陷的“挽救”重要的因素。