MDCK CELL MUTANTS DEFECTIVE IN GLYCOPROTEIN MATURATION
MDCK 细胞突变体糖蛋白成熟缺陷
基本信息
- 批准号:3436819
- 负责人:
- 金额:$ 10.02万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 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
项目摘要
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
项目成果
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