CLONING EXPRESSION AND TRANSPORT STUDIES OF SENDAI VIRUS

仙台病毒的克隆表达和转运研究

基本信息

批准号：
3436877
负责人：
NANCY Lugene MCQUEEN
金额：
$ 10.13万
依托单位：
CALIFORNIA STATE UNIVERSITY LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-05-01 至 1994-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3436877
关键词：
biological transport gene deletion mutation gene expression glycoproteins intracellular transport membrane proteins molecular cloning mutant nonhistone nucleoprotein parainfluenza virus type 1 protein biosynthesis protein sequence protein signal sequence protein transport virus envelope virus genetics virus protein

项目摘要

Polarized epithelial cells are important in regulating the environment between external and internal compartments in an organism. Many malignant and nonmalignant diseases are characterized by changes in cell polarity. It is not known whether these are a cause or effect of the specific disease, but an understanding of the biogenesis, transport, assembly and maintenance of polarized cells may lead to important insights on the mechanisms and/or treatments of these diseases. Studies providing the most insight into these processes have utilized, as models, the infection of polarized monolayers of MDCK cells by enveloped RNA viruses. Such studies have shown that certain viruses such as influenza virus and Sendai virus bud only from the apical domain, whereas other viruses such as vesicular stomatitis virus bud exclusively from the basolateral domain. This project proposes to clone, express and follow the transport in eukaryotic cells of proteins encoded in the viral genome of one of these viruses, Sendai virus, and its variant, Fl-R. The two viruses differ in their site of budding in polarized cells. In other viral systems, the envelope glycoproteins, when expressed without any other viral genes, have been shown to localize in the same domain from which the virus ultimately buds. This indicates that their polypeptide backbones contain a signal for polarized transport. We will first test to see if this holds true for the Sendai viral proteins. Assuming it does, we hope to utilize their difference in transport site to identify sorting signals in the membrane proteins. The differences in the RNA and protein sequences of the two viruses have recently been published. Utilizing this information we will use site specific mutagenesis to systematically change the variant sequences back to wild-type. Each possible combination of the wild-type and variant sequence will be expressed and the transport followed. Thus, we should be able to identify the mutation(s) responsible for the difference in polarized expression of the proteins.

偏振上皮细胞在调节环境方面很重要在生物体中的外部和内部隔室之间。许多恶性非恶性疾病的特征是细胞极性的变化。尚不清楚这些是特定的原因还是影响疾病，但了解生物发生，运输，组装和维持极化细胞可能会导致对这些疾病的机制和/或治疗。提供最多的研究对这些过程的了解，作为模型的感染。通过包裹的RNA病毒对MDCK细胞的偏振单层。这样的研究已经表明某些病毒，例如流感病毒和仙台病毒仅来自顶端域的芽，而其他病毒，例如水泡口腔炎病毒仅来自基底外侧结构域。这个项目提议克隆，表达和遵循真核细胞中的运输这些病毒之一，仙台病毒的病毒基因组中编码的蛋白质，及其变体Fl-R。两种病毒在其萌芽地点有所不同在极化细胞中。在其他病毒系统中，包膜糖蛋白，当没有任何其他病毒基因表达时，已显示出本地化在病毒最终芽的同一领域中。这表明它们的多肽骨架包含一个偏振运输的信号。我们将首先测试以查看仙台病毒蛋白是否正确。假设确实如此，我们希望利用它们在运输地点的差异识别膜蛋白中的排序信号。差异最近已经发表了这两种病毒的RNA和蛋白质序列。利用这些信息，我们将使用特定网站的诱变系统地将变体序列更改为野生型。每个野生型和变体序列的可能组合将是表达，然后运输。因此，我们应该能够确定导致极化表达差异的突变蛋白质。