Feng Hui, Zhang Yi-Bing, Zhang Qi-Min, Li Zhi, Zhang Qi-Ya, Gui Jian-Fang
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Wuhan 430072, China.
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Wuhan 430072, China
J Immunol. 2015 Feb 1;194(3):1225-38. doi: 10.4049/jimmunol.1402415. Epub 2014 Dec 22.
In mammals, type I IFNs (mainly IFN-α/β) are primarily regulated by transcription factors of the IFN regulatory factor (IRF) family. Fish IFNs do not show a one-to-one orthologous relationship with mammalian type I IFN homologues. Using a bacterial one-hybrid reporter screening system and an overexpression approach to explore the molecular mechanism underlying fish IFN induction, we identified zebrafish Danio rerio IRF (DrIRF)1 as a positive regulator of the fish IFN antiviral response. Among 12 zebrafish IRF family genes, DrIRF1 is most abundant in zebrafish immune tissues, including head kidney and spleen; upon virus infection, it is one of most significantly induced genes. Overexpression of DrIRF1 induces the expression of IFN and IFN-stimulated genes, hence protecting epithelioma papulosum cyprini cells against spring viremia of carp virus infection. As a transcription factor with constitutively nuclear retention, DrIRF1 directly binds to the IFN-stimulated regulatory element/IRF-binding element sites of zebrafish IFN promoters, which are dependent on four conserved amino acids of the N-terminal DNA-binding domain helix α3 motif. Mutation of either residue reveals a differential requirement for DrIRF1-mediated activation of zebrafish IFNϕ1 and IFNϕ3 promoters. Notably, C-terminal phosphorylation of DrIRF1 is observed and is not required for in vitro binding of DrIRF1 to fish IFN promoters. Unlike DrIRF3 and DrIRF7, which are responsible for differential expression of zebrafish IFNϕ1 and IFNϕ3 through the retinoic acid-inducible gene I-like receptor pathway, DrIRF1 works in concert with MyD88 to activate zebrafish IFNϕ3 but not IFNϕ1. These results provide insights into the evolving function of IRF1 as a positive IFN regulator.
在哺乳动物中，I型干扰素（主要是IFN-α/β）主要由干扰素调节因子（IRF）家族的转录因子调控。鱼类干扰素与哺乳动物I型干扰素同源物不存在一对一的直系同源关系。我们利用细菌单杂交报告筛选系统和过表达方法来探究鱼类干扰素诱导的分子机制，鉴定出斑马鱼Danio rerio IRF（DrIRF）1是鱼类干扰素抗病毒反应的正调控因子。在12个斑马鱼IRF家族基因中，DrIRF1在斑马鱼免疫组织（包括头肾和脾脏）中表达最为丰富；病毒感染后，它是诱导最为显著的基因之一。DrIRF1的过表达诱导干扰素和干扰素刺激基因的表达，从而保护鲤上皮瘤细胞免受鲤春病毒血症病毒感染。作为一种具有组成型核滞留的转录因子，DrIRF1直接结合斑马鱼干扰素启动子的干扰素刺激调节元件/IRF结合元件位点，这依赖于N端DNA结合结构域螺旋α3基序的四个保守氨基酸。任一残基的突变揭示了DrIRF1介导的斑马鱼IFNϕ1和IFNϕ3启动子激活的不同需求。值得注意的是，观察到DrIRF1的C端磷酸化，且其对DrIRF1在体外与鱼类干扰素启动子的结合并非必需。与通过视黄酸诱导基因I样受体途径负责斑马鱼IFNϕ1和IFNϕ3差异表达的DrIRF3和DrIRF7不同，DrIRF1与MyD88协同作用激活斑马鱼IFNϕ3而非IFNϕ1。这些结果为IRF1作为干扰素正调控因子的进化功能提供了见解。
