果蝇RNA沉默抗病毒作用机制及其微进化研究

The fruit fly Drosophila melanogaster is a powerful model for studying innate immunity. Recent studies have demonstrated that protection of Drosophila against viruses with an RNA genome is mainly mediated by the RNA interference (RNAi) pathway. There is observed difference in response to single stranded RNA virus (DCV) and double stranded RNA virus (DXV) in Drosophila. However little is known about the distinct genetic requirements for the small RNA-based immunity against DCV and DXV in adult flies. . We propose here to address these issues by infectivity assays and deep sequencing in wild type and RNAi-defective mutant flies challenged by viruses. The wild type and mutant flies will be inoculated with DCV and DXV respectively. We will study the difference in survival and the accumulated viral RNA levels over a time course. The total RNA will be isolated from the inoculated wild type flies and mutant flies at an appropriate post-inoculation time point, then both the small RNA library and RNA-seq library of each sample will be prepared and sequenced by high throughput sequencer. The viral siRNA pools and mRNA gene expression patterns from wild type flies and mutant flies inoculated with DCV and DXV will be investigated. Use of mutant flies combined with deep sequencing in this study will yield insights into key issues on RNA-based antiviral immunity, such as the distinct viral siRNAs biogenesis among wild type flies and mutant flies, distinct genetic requirements in response to DCV and DXV infection, the collaboration between RNA silencing and other antiviral mechanisms. We will determine the list of genes and long non-coding RNAs involved in antiviral immunity in Drosophila.. As fruit flies can be latently infected by DCV virus in nature, study of the adaptation between fruit flies and DCV virus provide important example of microevolution. We will study genetic variations in genome sequences of the fruit flies latently infected with DCV virus, especially the nucleotide variations of antiviral genes, which potentially leads to alternative function of proteins and differential gene expression. Investigation of the correlation among the population of viral small RNAs, the accumulated RNA levels of DCV virus, differenttial expression of antiviral genes and nucleotide variations of antiviral genes allows the nature of microevolutionary change to be characterized.

RNA沉默是果蝇抵抗外源病毒感染的主要机制，但果蝇针对单链和双链RNA病毒的免疫反应不同，其对应的分子机制仍不清楚。我们将以野生型果蝇和RNA沉默通路中相关基因的突变体为研究对象，分别接种这些基因型果蝇单链和双链RNA病毒后，研究它们的生存曲线和体内累积的病毒RNA量的相关性。我们将提取接种病毒后果蝇的总RNA，分别制备小RNA的cDNA文库和RNA-seq库，并进行深度测序。我们将通过对数据深度挖掘，来研究RNA沉默通路中各个基因抗病毒作用分子机制的差异，理解RNA沉默对单链和双链RNA病毒免疫作用差异的分子基础，建立RNA沉默抗病毒机制和其它抗病毒机制间的联系。我们还将研究果蝇的基因组变异，特别是引起抗病毒基因功能和表达水平改变的变异，和天然潜伏病毒之间互作的微进化关系。通过本课题的研究，将深入理解果蝇的RNA沉默抗病毒机制的分子基础、寄主和潜伏病毒互相适应的微进化关系等重要科学问题。

果蝇是研究天然免疫的良好模式生物、具有易饲养维护、世代时间短、遗传背景清晰、遗传资源丰富和研究手段多等优点。目前认为果蝇中RNA沉默是主要的抗病毒机制，但果蝇RNA沉默抗病毒机制研究并不充分，特别是自然感染果蝇的单链DCV病毒和双链DXV病毒。 果蝇接种DCV后导致果蝇死亡。 DCV病毒也能潜伏感染果蝇，果蝇体内的免疫基因的表达与病毒复制形成稳定的平衡关系。因此理解果蝇个体体内RNA沉默通路中各基因对DCV和DXV病毒作用的分子机制；研究导致果蝇的免疫基因表达谱差异的遗传机制具有重要科学意义。基于课题设定的研究目标，我们在研究内容上围绕“RNA沉默抗单链和双链RNA 病毒的分子机制研究”和“果蝇RNA沉默抗病毒机制的微进化研究”两个相互联系的子课题展开，并取得如下重要进展：1）RNA沉默抗单链和双链RNA病毒的分子机制研究。将DCV和DXV病毒分别注射dcr-2, loqs, r2d2和ago2果蝇突变体，这些果蝇突变体的存活率及体内检出的病毒含量存在明显差别；分离这些突变体果蝇的总RNA, 构建小RNA文库和RNA-seq文库，并进行深度测序。生物信息学分析表明DCV和DXV病毒的小RNA在这些突变体中的分布特性存在显著的差异。2）潜伏感染DCV果蝇的筛选和进化分析。对1000多株果蝇进行筛选鉴定, 获得潜伏感染DCV的果蝇18株。提取这些潜伏感染DCV果蝇的总RNA, 构建RNA-seq文库，进行深度测序。发现与病毒浓度呈正相关的基因主要与有丝分裂、雌性减数分裂及天然免疫反应有关。我们还发现在高浓度DCV病毒潜伏感染下， Jak-STAT通路的配体Upd2基因上调，而抑制Jak-STAT通路的SOCS基因下调，表明JAK-STAT通路可能参与DCV潜伏感染中宿主的抗病毒反应。我们还发展了一种生物信息学方法来评估和DCV含量正相关的信号通路，我们发现Wnt信号通路和FoxO信号通路在DCV潜伏感染的果蝇中分别被抑制和激活。3）鉴定发现新的抗病毒基因dip1。 我们通过S2细胞的体外敲除实验，证实了Dip1在细胞水平上具有抗病毒作用。通过对dip1基因突变果蝇分析，发现突变型果蝇对DCV病毒易感，并且这种高死亡率和果蝇体内的DCV病毒的复制量正相关。我们的结果加深了对果蝇RNA沉默抗DCV和DXV病毒的分子机制的了解。