IBDV拮抗细胞RNAi 的分子机制

Infection bursal disease virus(IBDV) causes severe immunosuppressive disease in young chickens and continues to pose a significant threat to the poultry. Our previous study has demonstrated that IBDV infection in vivo appeared to inhibit expression of MDA5 leading to the abnormal expression of IFNs.However, the involved molecular mechanism of these unusual reactions has not been clarified.. RNA interference(RNAi) is a part of antiviral innate defense response triggered by virus-specific dsRNA produced during infection. Recent studies have documented RNAi and IFN act in concert to prevent the virus replication in cells, and RNAi play a role upstream the IFN in the antiviral innate response. However, it is still unknown how the IBDV counteracts the antiviral innate defense response of RNAi and how MDA5 sensors dsRNA of virus to inhibit IFN signaling pathway to innitiate replication in eukaryotes.. To address these questions, we analyzes the profile of virus-derived siRNAs(vsiRNAs) in IBDV-infected DF-1 cells by small RNAs sequence， Northern blot and the anti-IBDV effects of vsiRNAs.To access the IBDV proteins influencing on RNAi, the luciferase reporter analysis is used to identify the virus-encoded suppressor of RNAi(VSRs). In addition, over-expression or decreased expression of VSRs in DF-1 cells will be applied to analyze the function during IBDV infection,by TCID50 detection the IBDV titer, mRNA and protein expression of virus by quantitative real time RT-PCR(qRT-PCR)and western blot,expression of virus-induced antiviral innate genes by qRT-PCR, the profile of virus-derived siRNAs(vsiRNAs).. Through analysis of the dynamic change relationship among the RNAi network, IBDV titer and antiviral innate immune genes, our results will provide direct experimental evidences to clarify the innate immune mechanisms of RNAi network and IFN network act in concert to prevent the virus replication in cells,which are very important for the establishment of effective novel approach for the control and prevention of IBD.

我们前期研究发现，IBDV 感染可抑制细胞MDA5和IFN的表达，其分子机制并不清楚。有研究表明，RNAi可影响IFN在细胞中发挥抗病毒作用，IBDV如何拮抗细胞的RNAi并影响MDA5介导的IFN信号通路的激活，其分子机制并不清楚。本项目拟用小RNA测序和Northern blot分析细胞针对IBDV感染产生的siRNA（vsiRNA）表达情况，并分析vsiRNA的抗IBDV感染作用；用双荧光素酶报告系统筛选拮抗细胞RNAi的病毒蛋白（病毒抑制子），分析病毒抑制子在IBDV感染中的作用，通过TCID50测IBDV滴度；用qRT-PCR和Western blot分析病毒各基因的mRNA和蛋白水平；用qRT-PCR分析抗病毒天然免疫基因的表达水平。综合分析细胞RNAi、IBDV滴度以及IFN等抗病毒基因之间的动态变化关系，揭开RNAi网络和IFN网络在细胞抗IBDV感染中的共同作用机制。

RNAi是真核生物中一种保守的抗病毒免疫方式，已在植物、线虫、真菌、昆虫和哺乳动物中发现其发挥着抗病毒作用，但RNAi是否在禽的细胞中发挥抗病毒免疫作用，尚不清楚。本研究以IBDV为模式病毒，我们首先通过高通量测序，证实了IBDV感染DF-1细胞会产生大量的来源于病毒基因组的vsiRNA，同时进一步用northern blot对表达丰度比较高的siRNA进行了验证。vsiRNA的发现表明，鸡的细胞中也存在RNAi现象。为了进一步分析，RNAi现象是否发挥抗病毒免疫的功能，我们用siRNA下调Dicer1和Ago2的转录，结果IBDV wt和rΔIBDV复制水平显著升高。为了分析IBDV是如何拮抗宿主的RNAi机制的，我们对IBDV的5个病毒蛋白拮抗宿主RNAi效果进行了分析，发现VP3具有VSR的功能，并且VP3可以通过与dsRNA结合来保护其不被降解。当VP3的2个功能域patch1和patch2被突变时，则可抑制其VSR功能。进行病毒拯救时，VSR缺陷的IBDV，其第1代病毒不能引起DF-1出现细胞病变，病毒滴度也不能测到，表明VSR缺陷，使IBDV的复制被显著抑制，当回补VSR时，比如FHV B2或IBDV VP3，则可恢复IBDV的复制能力。表明RNAi具有抗病毒活性。并且我们还证明，鸡的这种RNAi的抗病毒功能是不依赖于IFN通路。