MicroRNA靶向病毒携带的microRNA模拟靶序列对病毒的抑制作用

Micro RNA模拟靶序列(target mimic,TM)通过竞争性结合miRNA,从而干扰miRNA对靶标m RNA的调控.我们前期工作发现:以黄瓜花叶病毒(Cucumber mosaic virus,CMV)作为载体在植物体内表达TM序列有效地抑制了miRNA的活性或稳定性,从而消减了miRNA对靶基因的调控.但是,miRNA与CMV携带的TM序列的结合在一定程度上抑制了病毒的积累.研究分析了miRNA靶向病毒携带的TM序列对病毒抑制作用的内在原因.a.通过RNA印迹分析CMV携带不同miRNA TM序列对病毒积累的影响,进一步明确miRNA靶向病毒携带的TM序列对病毒的抑制作用;b.利用GFP作为报告基因,通过荧光显微镜、蛋白质印迹以及RNA印迹分析TM序列对重组病毒积累的影响;c.以GFP作为报告基因,利用荧光显微镜观察和免疫印迹方法分析模拟靶序列对GFP翻译的影响;d.利用CMV病毒的反式复制系统分析miRNA模拟靶序列对病毒负链RNA合成的影响.结果表明,多种植物内源的miRNA靶向CMV基因组携带的miRNA TM序列,在不同程度上抑制了病毒的积累,miRNA与其TM序列的结合抑制GFP蛋白的翻译和负链的合成.植物内源的miRNA通过与病毒基因组携带的miRNA模拟靶序列结合,通过抑制病毒蛋白的翻译以及病毒负链RNA的合成,从而降低了病毒的积累水平.基于该论文的研究结果有可能建立一种抗病毒的新方法.

MicroRNAs Targeting to Virus-carrying MicroRNA Target Mimics Inhibit Virus Accumulation

Background: MicroRNA Target Mimics (TM) interfere with regulation of miRNA to its target mRNA by competitively binding the miRNA. Previously, we found that Cucumber Mosaic Virus (CMV) as a vector expressing a TM sequence effectively inhibited the activity or stability of somemiRNAs in plants, thereby impaired their regulation to the targets. However, the miRNAs binding to their TM sequences carried by CMV inhibited accumulation of the virus to some extent. Objective: To analyze underlying reason of the inhibition to virus accumulation when a miRNA binds to its TM sequence in CMV genome. Method: Firstly, we analyzed influence of various miRNA TM sequences in CMV genome on virus accumulation using RNA blotting. Then, using GFP as a reporter gene, effect of the TM sequences on virus accumulation was analyzed by fluorescence microscopy, Western blotting and RNA blotting. Subsequently, using GFP as a reporter gene again, we analyzed effect of the TM sequences on GFP translation. Finally, the influence of the TM sequences on virus negative-strand RNA synthesis was tested using CMV trans-replication system. Result: Targeting of Five plant endogenous miRNA species tested to their TM sequences carried by CMV genome inhibited virus accumulation to a varied extent. Binding of the miRNAs to their TM sequences suppressed translation of the GFP protein and synthesis of the negative strand. Conclusion: Plant endogenous miRNAs targeting to their target mimics carried by viral genome inhibits translation of viral protein and synthesis of negative strand, thus reduces virus accumulation. The data present here provide possibility for developing a new antiviral methods.