细胞hnRNP M蛋白与病毒基因组相互作用对塞内卡病毒复制影响的研究  

The effects of cellular hnRNP M interaction with viral IRES on Senecavirus A replication

作　　者：曹志远 魏丹丹 王海伟[1] 孙超[1] 于力[1] CAO Zhi-yuan;WEI Dan-dan;WANG Hai-wei;SUN Chao;YU Li(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/牛羊传染病研究创新团队,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2022年第3期263-268,283,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31770173、32000126);国家重点研发项目(2016YFD0501505)。

摘　　要：为研究核不均一核糖核蛋白M(hnRNP M)在塞内卡病毒(SVA)复制过程中的作用,本研究构建了重组质粒pCAGGS-HA-hnRNP M,将该重组质粒转染至BHK-21细胞后经western blot检测,结果显示其可在细胞中正确表达hnRNP M蛋白。通过在BHK-21细胞中转染pCAGGS-HA-hnRNP M过表达或转染shRNA-hnRNP M敲低hnRNP M表达后感染SVA,利用western blot检测SVA结构蛋白VP2和病毒TCID50,分析hnRNP M对SVA复制的影响,结果显示,与对照组比较,过表达hnRNP M蛋白可极显著抑制SVA在BHK-21细胞中的复制(P<0.01),而敲低hnRNP M蛋白能极显著促进SVA在BHK-21细胞中的复制(P<0.01)。进一步利用RNA免疫共沉试验在SVA感染的BHK-21细胞中获得免疫沉淀复合物,提取其RNA后利用SVA基因组特异性引物进行PCR检测,结果显示宿主细胞hnRNP M蛋白和SVA基因组RNA存在相互作用。通过激光共聚焦试验检测SVA基因组和hnRNP M的定位情况,结果显示SVA感染宿主细胞后hnRNP M从细胞核转移至细胞质中,并与SVA基因组RNA在细胞质中共定位。通过双荧光素酶报告基因试验分别在过表达hnRNP M和敲低hnRNP M的细胞中检测SVA内部核糖体进入位点(IRES)的活性,结果显示过表达hnRNP M蛋白能够抑制SVA IRES介导的翻译活性,而下调hnRNP M蛋白的表达则能够促进IRES的活性。上述研究结果首次证实了宿主细胞蛋白hnRNP M与SVA基因组存在相互作用,其可通过抑制IRES的翻译活性进一步抑制SVA复制。本研究为深入研究SVA复制的分子调控机制奠定了基础。To investigate whether hnRNP M is involved in Senecavirus A(SVA)replication and infection,the recombinant plasmid PCAGGS-HA-hnRNP M was constructed in this study.After transfection into BHK-21 cells,the recombinant plasmid was detected by western blot,and the results showed that hnRNP M protein could be correctly expressed in cells.Subsequently,hnRNP M overexpression or knockdown of BHK-21 cells was infected with SVA,and the effect of hnRNP M on SVA replication was evaluated by Western blot and TCID50.The results showed that overexpression of hnRNP M significantly inhibited SVA replication in BHK-21 cells(P<0.01),while down-regulation of hnRNP M protein significantly promoted the replication of SVA in BHK-21 cells(P<0.01).Further,the immunoprecipitation complex was obtained from SVA-infected BHK-21 cells by RNA immuno-precipitation assay,and the RNA was extracted,reverse-transcribed and detected by PCR using SVA genome-specific primers.The RNA immunoprecipitation results showed an interaction between hnRNP M protein and SVA RNA in the case of SVA infection.Confocal laser microscopy detected the localization of the SVA genome and hnRNP M.The results showed that hnRNP M was transferred from the nucleus to the cytoplasm after SVA infection and co-localized with SVA RNA in the cytoplasm.In addition,hnRNP M overexpression of hnRNP M or knockdown of BHK-21 cells were detected by the dual-luciferase reporter assay to determine the SVA IRES activity.The dual-luciferase reporter assay showed that overexpression of hnRNP M protein inhibited IRES-mediated translation activity.In contrast,down-regulation of hnRNP M protein promoted IRES activity.This study demon-strated for the first time that host protein hnRNP M could interact with the SVA genome to affect the activity of SVA IRES and inhibit viral replication,laying a foundation to further understanding the molecular regulation mechanism of SVA replication in the host.

关 键 词：塞内卡病毒 内部核糖体进入位点 核不均一核糖核蛋白M 抑制作用 

分 类 号：S852.65[农业科学—基础兽医学]