核不均一核糖核蛋白K对塞内卡病毒IRES依赖性翻译及病毒复制影响的研究  

Heterogeneous nuclear ribonucleoprotein K participates in IRESdependent translation and virus replication of Senecavirus A

作　　者：张璐[1] 李露 李名洋 樊帅 何文瑞 张雨杭[1] 万博[1] 韩世充 ZHANG Lu;LI Lu;LI Ming-yang;FAN Shuai;HE Wen-rui;ZHANG Yu-hang;WAN Bo;HAN Shi-chong(International Joint Research Center of National Animal Immunology,College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[1]河南农业大学动物医学院动物免疫学国家国际联合研究中心,河南郑州450046

出　　处：《中国兽医科学》2023年第11期1415-1423,共9页Chinese Veterinary Science

基　　金：国家自然科学基金青年基金项目(32002278);中国博士后科学基金面上项目(2021M701104)。

摘　　要：基于前期塞内卡病毒(SVA)内部核糖体进入位点(internal ribosome entry site,IRES)的互作组学数据,本研究首先利用生物素标记RNA亲和捕捉技术和RNA免疫共沉淀试验,证实核不均一核糖核蛋白K(hnRNP K)与SVA基因组互作,且利用MTS试验证实敲降或过表达hnRNP K对细胞活性影响不显著。为深入探究hnRNP K在SVA复制过程中的功能,本研究利用Western-blot和TCID50试验检测hnRNP K蛋白对SVA病毒蛋白表达和病毒滴度的影响,结果显示,敲降hnRNP K显著降低病毒蛋白VP2的表达量及病毒滴度,而过表达hnRNP K则显著增加VP2表达量及病毒滴度。利用间接免疫荧光和核质分离试验观察病毒感染对hnRNP K亚细胞定位的影响,发现SVA感染可诱导hnRNP K由细胞核向细胞质移位。通过双荧光素酶报告基因试验检测hnRNP K对帽依赖性翻译和SVA IRES依赖性翻译活性的影响,结果表明,敲降和过表达hnRNP K蛋白均对帽依赖性翻译活性无显著影响,敲降hnRNP K显著降低SVA IRES依赖性翻译活性,而过表达hnRNP K则显著增强IRES翻译活性。综上所述,本研究证实hnRNP K是SVA的关键反式作用因子,正调控病毒IRES依赖性翻译及病毒复制,为深入了解小RNA病毒蛋白翻译和复制的分子机制提供了新的见解。Based on the previous interactome analyses of host factors interacting with the internal ribosome entry site(IRES)of Senecavirus A(SVA),we first confirmed that hnRNP K interacted with the SVA IRES by biotinylated RNA pulldown assay and RNA-protein immunoprecipitation in this study.We also demonstrated that knockdown or ectopic expression of hnRNP K had negligible effects on cell viability by MTS assay.To further determine the functional role of hnRNP K in the SVA replication cycle,we investigated the effect of hnRNP K knockdown or overexpression on viral protein expression and virus titers by western blot and on TCID50,respectively.The results showed that knockdown of hnRNP K significantly reduced the expression of viral protein VP2 and virus titers,while overexpression of hnRNP K had opposite effects on the expression of VP2 and virus titers.Subsequently,we observed that hnRNP K displays a dramatic re-localization from the nucleus to the cytoplasm of the cell in response to SVA infection by confocal immunofluorescence microscopy analysis and nuclear-cytoplasmic fractionation analysis.Finally,by using a bicistronic luciferase construct psiCHECK-SVA and dual-luciferase reporter assay,we found that cellular cap-dependent translation activity was not affected when hnRNP K was knocked down or ec topically expressed.However,viral IRES-dependent translation activity was significantly reduced by the depletion of hnRNP K and greatly elevated by the ectopic expression of hnRNP K.Taken together,this study demonstrated that hnRNP K acts as a crucial trans-acting factor that positively regulates viral IRES-dependent translation and replication of SVA,providing novel insights on further understanding the molecular mechanism of picornavirus replication.

关 键 词：核不均一核糖核蛋白K 塞内卡病毒 内部核糖体进入位点 核质移位 

分 类 号：S852.659.6[农业科学—基础兽医学]