传染性法氏囊病毒对靶细胞泛素－蛋白酶体通路的干扰机制

项目名称： 传染性法氏囊病毒对靶细胞泛素－蛋白酶体通路的干扰机制

项目编号： No.30870117

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 周继勇

作者单位： 浙江大学

项目金额： 40万元

中文摘要： 本项目以合成多肽为抗原制备了兔抗UPP相关蛋白的多克隆抗体，通过双免疫荧光标记结合免疫共沉淀技术，证明IBDV编码的VP3与泛素蛋白及E2存在相互作用关系，提示IBDV VP3可能是通过宿主UPP的这条途径进行降解的。荧光定量RT-PCR分析显示，在IBDV感染细胞与VP3重组载体转染细胞中，其UPP相关基因及UPP介导的细胞凋亡信号通路因子的转录本变化趋势相似，提示VP3与UPP的互作在IBDV感染细胞过程中发挥重要作用。采用Western blot法对UPP通路、Caspase途径、MHC途径和NF-κ#36884;径相关组分的蛋白水平进行验证。蛋白酶体水解功能分析显示，IBDV VP3不影响宿主蛋白酶体水解活性。采用MG132抑制蛋白酶体活性后，不同程度地抑制了病毒基因复制、病毒蛋白表达、病毒产量、病毒释放和增殖能力。这些结果提示，UPP在IBDV复制过程中发挥重要作用。

中文关键词： 传染性法氏囊病病毒；泛素蛋白酶体通路；相互作用；干扰机制

英文摘要： In this projects, we prepared rabbit pAb to UPP-associated proteins, and confirmed the interactions between VP3 and ubiquitin/E2 by combining the dual-staining immunofluorescence and coimmunoprecipitation methods, indicating that IBDV VP3 may be degraded by host UPP.The transcriptional profiles of UPP-associated genes and cellular signaling factors involved in apoptosis in IBDV-infected and VP3-transfected cells were determined by real-time RT-PCR, same results suggested that interaction between VP3 and UPP play an important role in IBDV infection. Using Western blot, we further analyze the expression profiles of cellular proteins involved in UPP, caspase pathway, MHC pathway and NF-KB pathway. Protelytic analysis showed that IBDV VP3 does not change the protelytic activity of host proteasome. Using the drug MG132 to inhibit the activity of proteasome, the viral gene replication, viral expression, viral production and viral release were largely inhibited, suggesting that UPP play an essential role during IBDV infection.

英文关键词： infectious bursal disease virus; ubiquitin-proteasome pathway; interaction; interference mechanism