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Characterization of a CD99-related 21 -kDa cellular protein(VAP21) incorporated into rabies virion

掺入狂犬病毒颗粒中的 CD99 相关 21 kDa 细胞蛋白 (VAP21) 的表征

基本信息

批准号：
12670281
负责人：
TOCHIKURA Tadafumi
金额：
$ 1.47万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670281/
关键词：
Rabies virus Vesicular stomatitis virus VAP21 CD99 Syrian hamster BHK-21 Tetracycline-regulated gene expression system G418 狂犬病ウイルス宿主由来成分 pTet-Splice アンチセンス

项目摘要

The VAP21, a CD99-related 21-kDa cellular transmembrane protein, was expressed in various types of organs of Syrian hamster as well as the hamster-derived cell lines (BHK-21 and HmLu-1). The protein displayed heterogeneous electrophoretic mobilities depending on the organs and cell lines probably due to different posttranslational modifications. The VAP21, however, was not expressed in other animal species we tested, including Chinese hamster, mouse, dog, monkey and human. We tried to express the VAP21 in VAP21-negative cell lines, such as CHO-K1 , HeLa, COS-7 and MDCK, using a tetracycline-regulated gene expression system. All of the trials, however, resulted in obtaining no permanently positive nor inducible cell lines : at the beginning of the cDNA transfection, cells were transiently positive for VAP21 expression, but the positive population decreased to undetectable levels within a few weeks, even though not a few G418-resistant clones were obtained. To the contrary, we could easily establish the VAP21-overexpressing cell lines from Syrian hamster cells, which were successfully maintained without any loss of VAP21 expression even under the induced culture conditions. Furthermore, although transfection of the VAP21 cDNA affected the recipient cells differently depending on the cell types, the VAP21 expression was somewhat of benefit to infection with the vesicular stomatitis virus as seen in the increased yield of progeny viruses. Interestingly, although VAP21 antigen was diffusely distributed as very fine dots over the entire cell surface of BHK-21 and HmLu-1 cells, alteration of the distribution pattern such as the development of bleb-shaped spots on the cell surface was observed both in BHK-21/VAP21 and HmLu-1/VAP21 cells. These results provide important implications for potential role of VAP21 in cellular function.

VAP 21是一种CD 99相关的21-kDa细胞跨膜蛋白，在叙利亚仓鼠的各种类型的器官以及仓鼠衍生的细胞系（BHK-21和HmLu-1）中表达。该蛋白质显示异质性电泳迁移率取决于器官和细胞系可能是由于不同的翻译后修饰。然而，VAP 21在我们测试的其他动物物种中不表达，包括中国仓鼠、小鼠、狗、猴和人。我们尝试使用四环素调控的基因表达系统在VAP 21阴性的细胞系中表达VAP 21，如CHO-K1、HeLa、COS-7和MDCK。然而，所有的试验都没有获得永久阳性或可诱导的细胞系：在cDNA转染开始时，细胞对VAP 21表达呈瞬时阳性，但阳性群体在几周内下降到不可检测的水平，即使获得了不少G418抗性克隆。相反，我们可以容易地从叙利亚仓鼠细胞中建立VAP 21过表达细胞系，即使在诱导培养条件下，该细胞系也成功地维持而没有VAP 21表达的任何损失。此外，尽管VAP 21 cDNA的转染对受体细胞的影响因细胞类型而异，但VAP 21的表达对水泡性口炎病毒的感染有些益处，如在子代病毒产量增加中所见。有趣的是，尽管VAP 21抗原作为非常细的点弥散分布在BHK-21和HmLu-1细胞的整个细胞表面上，但在BHK-21/VAP 21和HmLu-1/VAP 21细胞中均观察到分布模式的改变，例如在细胞表面上形成水泡状斑点。这些结果为VAP 21在细胞功能中的潜在作用提供了重要的启示。