20S蛋白酶体亚基在脑心肌炎病毒复制和组装中的作用

项目名称： 20S蛋白酶体亚基在脑心肌炎病毒复制和组装中的作用

项目编号： No.31300139

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 生物科学

项目作者： 黄丽

作者单位： 中国农业科学院哈尔滨兽医研究所

项目金额： 23万元

中文摘要： 脑心肌炎病毒（EMCV）是一种重要的人畜共患病病原，该病毒利用宿主蛋白完成自我复制和组装，但宿主蛋白如何参与EMCV的复制和组装过程还不是很清楚。我们首次研究发现，20S蛋白酶体的多个亚基被包装入EMCV病毒粒子。目前，20S蛋白酶体及其亚基参与EMCV的生命周期的研究还没有报道。本项目将通过特异的蛋白酶体抑制剂抑制20S蛋白酶体的活性、过度表达或RNA干扰下调20S蛋白酶体亚基蛋白表达等方法研究20S蛋白酶体亚基对EMCV复制的影响。同时利用酵母双杂交、免疫共沉淀等方法筛选与20S蛋白酶体亚基相互作用的病毒蛋白，研究20S蛋白酶体亚基如何与EMCV基因组或编码蛋白相互作用，阐明其包装入病毒粒子的分子机制。本研究对阐明EMCV复制及病毒粒子组装机制具有重要意义，也为以后研究其它宿主蛋白在EMCV复制过程中的作用打下坚实基础。

中文关键词： 脑心肌炎病毒；蛋白酶体亚基；3C蛋白酶；切割；TANK

英文摘要： The Encephalomyocarditis virus is a potential zoonotic agent. Host proteins are required for EMCV life cycle. Until now，few host proteins involved in EMCV replication and assembly were identified and the molecular mechanism of EMCV life cycle is unknown. Our previous studies showed that several subunits of 20S proteasome were incorporated into EMCV viron by using liquid chromatograph mass spectrometry method (LC-MS). These 20S proteasome subunits include PMSA1, PMSA5, PMSA6, PSMB2, PMSB4, PMSB5 and PMSB6. However, the functions of these subunits of 20S proteasome in EMCV infection, replication and assembly are unknown. In this project,(1)we will study the functional roles of 20S proteasome subunits in EMCV replication after the BHK-21 cells are treated with specific proteasome inhibitors. Meanwhile, EMCV replication and growth will be studied after knockdown or overexpression of these 20S proteasome subunits in BHK-21 cells.(2) We will investigate whether EMCV genomic RNA interacts with these 20S proteasome subunits by using RNA-pulldown to test whether EMCV genomic RNA is required for 20S proteasome subunits to assembly into EMCV virions. (3) We will identify the interactions between EMCV viral proteins and 20S proteasome subunits by using yeast two-hybrid (Y2H ) and Co-immunoprecipitation (Co-IP). 　　 Ou

英文关键词： Encephalomyocarditis virus；Proteasome subunit；3C protease；cleave；TANK