黑鲷JAK-STAT信号通路在鱼类神经坏死病毒感染过程中的作用机制研究

Aquaculture has made an enormous contribution to the world food production, especially to the sustainable supply of animal proteins. China has the largest quantity of the aquiculture products in the world, and aquiculture plays a dominant role in fishery in China. However, the frequent outbreaks of viral and bacterial diseases have caused severe economic losses, and disease has become the key factor restricting sustainable development of fishies. Interferon (IFN) response is the first and primary line of host defense against virus infection in teleost. IFN signaling activates the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway, leading to transcription of IFN-stimulated genes (ISGs), which help host establish antiviral status to defect virus replications. IFN bind a heterodimeric transmembrane receptor termed the IFNα receptor(IFNAR), which is composed of IFNAR1 and IFNAR2 subunits. IFNAR engagement was shown to activate the receptor-associated protein tyrosine kinases Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2), which phosphorylate the latent cytoplasmic transcription factors signal transducer and activator of transcription 1 (STAT1) and STAT2. Tyrosine-phosphorylated STAT1 and STAT2 dimerize and translocate to the nucleus, where they assemble with IFN-regulatory factor 9 (IRF9) to form a trimolecular complex called IFN-stimulated gene factor 3 (ISGF3). ISGF3 binds to its cognate DNA sequences, which are known as IFN-stimulated response elements (ISREs), thereby directly activating the transcription of ISGs. This is the classic JAK-STAT signaling pathway. Recently, based on efforts of scientist, a great attempt to understanding of fish IFN response provides evidence that fish appear to trigger IFN antiviral response by the similar mechanisms to that in mammals. However, the molecular mechanisms of IFN inducing and activating JAK-STAT signaling pathways to protect the host from NNV infection was still unclear in teleost fish. Nervous necrosis virus (NNV) can cause mass mortalities of many marine and freshwater fishes in the world, the mortality of larvae and juvenile were usually about 90% during disease outbreak. NNV belongs to the betanodaviruses of nodaviridae and is a nonenveloped icosahedral particle with 2 single-stranded positive-sense RNAs. Our previous study showed that black seabream RLR receptors could recognize NNV, induce IFN production, activates JAK-STAT signaling pathways to establish antivirus status. In this study, we performed a comprehensive analysis of the functions of several crucial factors in JAK-STAT signaling pathway including STAT1, STAT2 and IRF9, and their phosphorylation mechanism during infection of NNV. Based on the above work, we will further uncover the regulation mechanisms of ISGs transcription during NNV function. Our study will highlight the importance of JAK-STAT signaling pathway in host defense and provide insight on the mechanisms of innate immune responses following NNV infection. This research will provide the theory basis for further clarifying the infection mechanism of NNV, immune evasion strategies and antiviral drug development.

我国是水产养殖大国，但病害是水产养殖的重要威胁。黑鲷是名贵海水养殖鱼类，幼苗易受神经坏死病毒(NNV)感染。研究表明干扰素(IFN)通过JAK-STAT信号通路启动抗病毒ISG基因的表达，是脊椎动物抵御病毒入侵的第一道防线。我们的前期结果显示黑鲷RLR受体能识别NNV诱导产生IFN激活JAK-STAT信号通路，表明黑鲷JAK-STAT信号通路在抗NNV感染过程中发挥重要作用，但该通路中的关键基因STAT1/STAT2/IRF9的功能和作用机制尚不清楚。本课题拟在此基础上通过基因缺失突变、免疫共沉淀和启动子活性分析等技术手段来研究黑鲷STAT1、STAT2和IRF9的功能，及其调控下游ISG基因的机制，来阐明黑鲷JAK-STAT信号通路抗NNV感染的分子作用机制，这将为鱼类预防NNV和抗NNV药物的研发提供理论基础。

我国是水产养殖大国，但病害是水产养殖的重要威胁。神经坏死病毒(NNV)是一种世界范围内流行的传染性病原，能感染120多种海淡水经济鱼类的种苗和部分成鱼，造成巨大经济损失。干扰素(IFN)通过JAK-STAT信号通路启动抗病毒ISG基因的表达，是脊椎动物抵御病毒入侵的第一道防线。黑鲷是名贵海水养殖鱼类，幼苗易受神经坏死病毒(NNV)感染而大量死亡。前期结果显示RLR受体能识别NNV诱导产生IFN激活JAK-STAT信号通路，表明黑鲷JAK-STAT信号通路可能在抗NNV感染过程中发挥重要作用。本项目对黑鲷JAK-STAT信号通路在神经坏死病毒感染过程中的作用机制进行研究。通过原代细胞培养建立了对NNV敏感的黑鲷脑细胞和脾脏细胞系，构建了NNV体外研究平台。在该平台的基础上，我们克隆了一批与黑鲷JAK-STAT信号通路的相关基因，并通过体内外病毒感染和表达分析，证明RGNNV感染能够激活黑鲷JAK-STAT信号通路。进一步对黑鲷JAK-STAT信号通路的激活、信号传递和抗病作用进行了分析。我们的研究表明黑鲷IFN-γ能够快速激活JAK-STAT信号通路，从而促进下游抗病基因的表达，抑制NNV的复制和产生。我们研究了黑鲷IRF9的亚细胞定位机制，结果表明与其他IRF9不同，黑鲷IRF9不仅包含经典的N端核定位信号，其C端还存在其他的核定位信号。我们探索了黑鲷JAK-STAT信号通路下游抗病基因ISG15的表达、功能和转录调控机制：黑鲷ISG15的抗病毒作用依赖于C末端LRGG的泛素化修饰序列；鉴定了黑鲷ISG15启动子的核心ISRE位点；发现ISG15启动子附近的内含子序列通过GAS位点增强黑鲷ISG15转录。通过对黑鲷ISG15转录核心位点ISRE和GAS的鉴定，证明JAK-STAT信号通路核心复合物ISGF3在下游抗病毒ISG的转录激活过程中发挥关键作用。上述研究为阐明鱼类JAK-STAT在抗病天然免疫过程中的分子机制奠定了基础，为鱼类预防NNV和抗NNV药物的研发提供理论基础。