宿主microRNA抑制流感病毒复制的机制研究

Antigenic shift and drift occur in influenza A virus frequently and usually cause pandemics. Influenza epidemics and pandemics have a huge impact on society and individuals all over the world. The replication of influenza virus is both positively and negatively regulated by host factors. It is reported that the host cell microRNAome were changed during influenza virus infection which implied that some of microRNAs are involved in influenza virus replication and may play important roles in the process of virus infection and host immunologic defense. Our preliminary data showed that miR-125a，miR-30d，miR-181a，miR-27a，miR-33a and miR-194 elicit significant inhibitory effect on the replication of influenza virus. In order to explore the mechanism of these miRNAs on regulating influenza virus replication, we proposed the following specific aims: (1) To investigate whether these microRNAs directly target influenza virus genes by taking the approach of 3'UTR reporter assay. (2) To study whether the microRNAs activate the host immune response by targeting host genes. (3) To understand if the influenza virus evovled the stratagem to avoid the inhibition by host microRNAs . (4) To determine the antiviral effect of microRNA in vivo by using synthetic miRNA mimics and mouse infection model. These studies will explore the mechanism of how the host miRNAs involved in virus replication and provide a new idea for discovering new anti-flu drugs.

流感病毒易发生变异，能引起世界范围的流感大流行，给人类健康带来了极大的危害。研究表明流感病毒的复制需要宿主因子的辅助，同时亦受到宿主因子的抑制。在流感病毒感染过程中，宿主细胞中microRNA的表达谱会发生显著的变化，我们对部分差异表达的microRNA进行了初步筛选，发现6个候选microRNA对流感病毒具有显著的抑制作用，因此本项目拟在此基础上进行以下几方面的研究：（1）通过荧光素酶报告基因分析的方法，分析上述microRNA是否直接靶向流感病毒基因，并定位它们在病毒基因组上的作用靶点；（2）通过反向遗传方法构建流感病毒突变株并通过同源性比对研究病毒的逃逸机制；（3）分析microRNA是否通过靶向宿主mRNA，激活宿主抗病毒反应，从而调控病毒的复制；（4）以小鼠为动物模型，研究相应的microRNA模拟物的抑病毒功能，为抗流感病毒药物的研发提供新思路。

本研究采用定量RT-PCR、免疫斑点杂交、荧光素报告检测等分子生物学技术，以及激光共聚焦、流式细胞术等手段在细胞水平，首次发现：（1）miR-33a等6条miRNA可显著抑制H1N1型流感病毒在宿主细胞中的复制；（2）miR-33a对H3N2和H9N2流感病毒在细胞中的复制也有显著的抑制作用；（3）miR-33a可通过直接靶向ARCN1 mRNA的3’UTR区，从而抑制ARCN1的蛋白水平表达；（4）miR-33a不抑制流感病毒与宿主细胞膜的结合，但可通过抑制ARCN1的表达，从而抑制流感病毒与宿主细胞膜的融合过程；（5）miR-33a可抑制流感病毒核蛋白复合体的活性；（6）miR-181a和miR-181b可通过直接靶向KPNB1 mRNA 3’UTR而抑制其表达。进而抑制流感病毒NP蛋白向细胞核的转运；（7）通过基于PABPC1的RNA免疫共沉淀实验，可富集靶向单一mRNA的miRNA集合。上述结果充分说明，作为细胞重要的调节分子，miRNA在宿主的抗病毒感染过程中起着重要的作用。同时，要从整体水平研究宿主miRNA对病毒mRNA翻译过程的调控过程，可采用针对PABPC1的RNA免疫共沉淀技术。