文章摘要
草鱼细胞蛋白酶亚基β7与草鱼呼肠孤病毒非结构蛋白NS12相互作用
Cellular proteasome subunit beta type-7 interacts with grass carp reovirus non-structural protein 12
投稿时间:2021-01-12  修订日期:2021-03-05
DOI:
中文关键词: 草鱼呼肠孤病毒  NS12  PSMB7  蛋白酶  相互作用
英文关键词: GCRV  NS12  PSMB7  Proreasome  Interaction
基金项目:现代农业产业技术体系建设专项资金(CARS-45-19)
作者单位邮编
阙顺政 上海海洋大学 201306
王龙龙 上海海洋大学 
吕利群 上海海洋大学 201306
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中文摘要:
      蛋白酶体亚基β7(PSMB7)是20S核心蛋白酶体复合物的主要组成部分之一,具有类胰蛋白酶的活性。草鱼呼肠孤病毒(Grass carp reovirus, GCRV)外衣壳蛋白VP7已证实与其在体外和体内存在着相互作用。我们通过酵母双杂交实验表明PSMB7与GCRV编码的膜相关的非结构蛋白NS12也存在着潜在相互作用。为了证实两者之间的相互作用,我们进行了GST-Pulldown实验,明确GST标签的NS12与GFP标签的PSMB7存在相互作用。在GCRV感染草鱼GCO细胞过程中,我们观察到PSMB7在细胞中的表达水平恒定。在GCRV感染过表达PSMB7的GCO细胞后进行real time RT-PCR检测,发现PSMB7的过表达有利于病毒RNA的转录。蛋白免疫印迹结果显示,NS12对蛋白降解并不敏感。综上所述,这些结果表明GCRV NS12与PSMB7存在分子间相互作用,但并没有作为PSMB7的底物。表明其可能竞争性地阻碍蛋白酶体复合物的形成。病毒蛋白干扰蛋白酶体复合物胞内PSMB7的积累可能是其一种针对蛋白酶体介导的先天免疫的病毒策略。
英文摘要:
      Proteasome subunit beta type-7 (PSMB7) is one of the major components of the 20S core proteasome complex and displays a trypsin-like activity. Capsid protein VP7 of grass carp reovirus (GCRV) has been shown to interact with PSMB7 both in vitro and in vivo. Here, yeast two-hybrid screening revealed that grass carp PSMB7 interacted with NS12 protein, a membrane-associated protein encoded by GCRV. To confirm the physical interaction between PSMB7 and NS12, we performed pull down analysis to demonstrate the physical association between GST-tagged NS12 and GFP-tagged PSMB7. Constant expression level of PSMB7 in grass carp GCO cells during GCRV infection course was observed, and over-expression of PSMB7 seemed to favor viral RNA transcription in our transfection and infection assays. NS12 expressed during viral infection or plasmid transfection was not susceptible to proteomic degradation in Western blot analysis. Taken together, these results indicated that GCRV NS12 interacted with PSMB7, but didn’t serve as a substrate for PSMB7, suggesting its potential role in competitively impeding the formation of proteasome complex. Interfering with the accumulation of intracellular PSMB7 in proteasome complex by viral proteins seemed to represent a novel viral strategy against the proteasome-mediated innate immunity.
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