宿主蛋白核仁素与病毒基因组相互作用对牛肠道病毒复制影响的研究

Cellular nucleolin interacts with the viral genome to promote the replication of bovine enterovirus

为了探究宿主蛋白核仁素(NCL)对牛肠道病毒(BEV)复制的影响及其作用机制,本研究将构建的重组质粒pHA-NCL和NCL shRNA分别转染BHK-21细胞,采用western blot检测细胞中NCL的表达。结果显示,NCL在BHK-21细胞中分别获得了过表达和下调表达。在此基础上,将BEV以MOI 1感染上述过表达和下调NCL表达的BHK-21细胞,于感染8 h、10 h和12 h时收集上清液和细胞,采用qPCR、western blot(检细胞)分别检测病毒基因组RNA的复制水平、BEV结构蛋白VP1表达水平及通过上清液检测病毒滴度的变化。结果表明,过表达NCL明显促进BEV在BHK-21细胞中的复制,而下调NCL的表达则明显抑制BEV的复制。利用兔源NCL特异性抗体及同源IgG对感染BEV的BHK-21细胞进行RNA免疫共沉淀试验,并利用RT-PCR检测免疫复合物中BEV相应基因片段。结果显示,免疫复合物NCL抗体-NCL蛋白-病毒RNA免疫复合物中扩增出BEV目的基因片段,而IgG抗体对照组未扩增出任何条带,表明NCL与BEV基因组RNA存在相互作用。通过激光共聚焦试验观察BEV感染BHK-21细胞后不同时间(5 h和10 h)NCL蛋白的亚细胞定位,结果显示,BEV感染细胞后使NCL由核仁迁移至细胞质,并与病毒基因组在细胞质中共定位。利用同源重组试剂盒将线性化的pmirGLO及扩增的BEV内部核糖体进入位点(IRES)构建双荧光素酶报告质粒pmirGLO-BEV IRES,并经测序鉴定正确后分别转染NCL过表达和下调表达的BHK-21细胞,采用双荧光素酶试剂盒分别检测上述细胞中两种荧光素酶FLuc和RLuc(BEV IRES依赖性的)的活性。结果显示,与转染pHA的对照细胞相比,NCL过表达的BHK-21细胞中FLuc值无显著变化,而RLuc值显著升高(P<0.01);与转染shRNA NC的对照细胞相比,NCL表达下调的BHK-21细胞中,FLuc值无显著变化,而RLuc值显著降低(P<0.01)。表明,NCL正调控BEV IRES的翻译起始活性。本研究首次证实NCL与BEV基因组间存在相互作用,该互作可促进BEV IRES介导的病毒蛋白翻译活性,从而提高BEV的复制水平,为BEV复制的分子调控机制提供了新的见解。

In order to investigate the effect of cellular nucleolin(NCL)on bovine enterovirus(BEV)replication and its mechanism,the constructed recombinant plasmid pHA-NCL and NCL-targeting shRNA were transfected into BHK-21 cells,respectively.Western blot was used to detect the expression of NCL in cells.The results showed that NCL was overexpressed or down-regulated in BHK-21 cells.On this basis,the above-mentioned NCL overexpressed or down-regulated BHK-21 cells were infected with BEV at MOI of 1.The supernatant and cells were collected at 8 hours,10 hours and 12 hours after infection.RT-qPCR and western blot(to detect cells)as well as TCID50(to detect supernatant)were used to detect the replication of viral genomic RNA,the expression level of BEV structural protein VP1 and the changes of virus titers.Overexpression of NCL significantly promoted BEV replication in BHK-21 cells,while down-regulation of NCL significantly inhibited BEV replication.The RNA immunoprecipitation was performed in BHK-21 cells infected with BEV using rabbit NCL-specific antibody and homologous IgG,and RT-PCR amplified the gene fragment of BEV.The results showed that the immune complex containing the NCL antibody amplified the BEV target gene fragment,while the IgG control group failed to amplify any band,indicating that NCL interacted with BEV genomic RNA.The subcellular localization of NCL protein at different times(5 hours and 10 hours)after BEV infection of BHK-21 cells were observed by confocal assay.The subcellular localization of NCL in BHK-21 cells was observed at 5 hours and 10 hours after BEV infection.The results showed that NCL shuttled from the nucleoli to the cytoplasm after BEV infection,and co-located with the viral genome in the cytoplasm.The linearized pmirGLO and the amplified internal ribosome entry site(IRES)of BEV were used to construct the bicistronic reporter plasmid pmirGLO-BEV IRES by homologous recombination kit,and were identified by sequencing and transfected into BHK-21 cells with overexpression and down-regulation of NCL,the expression of FLuc and RLuc(BEV IRES-dependent)were detected in the above cells by double fluorescein reporting kit.No significance for the FLuc expression in NCLoverexpressing BHK-21 cells was observed when compared with pHA-transfected control cells,while the RLuc overexpression was significantly increased(P<0.01).In contrast,the FLuc expression in BHK-21 cells with NCL down-regulated expression showed no significance when compared with NC-transfected control cells,while RLuc overexpression was significantly decreased(P<0.01).It was shown that overexpression of NCL significantly enhanced while down-regulation significantly inhibited the translation initiation activity of BEV IRES,respectively.In this study,we demonstrated for the first time that NCL interacts with the BEV genome,thus promoting BEV IRES-mediated viral protein translation.We provide new insights into the molecular regulation mechanism of BEV replication.