鱼类特有基因Gig1和Gig2在干扰素抗病毒反应中的功能研究

Curcian carp Carassius autuas Gig1 and Gig2 (grass carp reovirus-induced gene 1 and 2) are recently identified as two novel genes that are significantly induced in viral-infected cells and found exclusively in fish genomes. Based on previous studies on gene expression, Gig1 and Gig2 seem to be two representatives of host interferon stimulated genes (ISGs), one being an early phase ISG and the other being a late phase ISG; however, the detailed mechanism underlying both gene expression by virus infection and the exact physiological roles of both genes responsible for interferon antiviral responses remain to be investigated. In this project, first, the 5' flanking sequences of both genes will be cloned to compare their induction pathways triggered by virus infection, thus clarifying how both genes are transcriptionally iniated during interferon antiviral responses. Second, the dynmics of subcellular localization of both gene-encoding proteins will be studied in order to reveal where both genes function in viral-infected fish cells, respectively. Considering that ISGs might act as antiviral effectors to shut down virus resplication or important factors to influence the production of IFN,finally, we will try to clarify whether both genes might directly inhibit virus propagation or participate in regulation of IFN expression or act both.If this poject is supported, the data will be made to clarify how, where and what Gig1 and Gig2 function, and importanly, to aid in understanding of fish own interferon-mediated antiviral mechanims triggered by fish-specific genes.

鲫Gig1和Gig2是两个最近鉴定的、在病毒感染的细胞中大量表达的新基因，目前仅在鱼类基因组中发现有同源基因存在。前期研究表明Gig1和Gig2可能代表干扰素诱导的早期基因和晚期基因。然而，其中的表达调控机制以及这两个基因的生理功能都不曾进行过深入研究。本项目首先将分离鉴定Gig1和Gig2基因的侧翼调控序列，比较研究病毒诱导Gig1和Gig2的表达调控，揭示Gig1和Gig2在病毒感染的细胞中如何被启动表达。进而研究Gig1和Gig2基因编码蛋白在病毒感染细胞中的动态定位规律，揭示它们在干扰素抗病毒反应中发挥功能的场所。最后从抗病毒作用和调控干扰素反应信号通路两个方面对Gig1和Gig2的可能功能进行探索研究。本项目的研究结果不仅将阐明Gig1和Gig2在干扰素抗病毒免疫反应中的功能角色以及作用机理，而且有助于理解鱼类具有自身特点的干扰素抗病毒反应机制。

鲫Gig1和Gig2是我们实验室在2004年从用紫外线灭活GCRV感染的鲫培养细胞中发现的受病毒诱导表达的新基因。本项目的研究结果显示：鱼类Gig1和Gig2基因不仅受病毒诱导表达，而且受poly(I:C)等干扰素诱导剂诱导表达。这两个基因被诱导表达的分子机制是：细胞先通过RLR介导的信号通路诱导宿主细胞产生干扰素，干扰素再通过Jak-Stat信号通路诱导Gig1和Gig2基因的表达。启动子分析支持Gig1和Gig2是通过RLR-IFN途径被诱导表达的结论。亚细胞定位分析揭示Gig1和Gig2是两个定位在细胞质中的蛋白，即使在病毒感染时也定位在细胞质中。过量表达Gig1和Gig2促进细胞存活、抑制GCRV和SVCV等病毒在细胞中的复制。这些结果表明鱼类Gig1和Gig2是两个由干扰素诱导表达的抗病毒蛋白，在细胞质中发挥抗病毒功能。进一步的分子机制研究揭示鱼类Gig1可能通过正调控RLR和TLR途径中的重要信号分子和某些抗病毒蛋白的表达发挥抗病毒作用。最后，研究发现Gig1和Gig2在鱼类具有多拷贝的同源基因，Gig1特异存在于硬骨鱼类基因组中，而Gig2广泛存在于低等脊椎动物包括从无颌动物（如七鳃鳗）到两栖类的物种基因组中，而在羊膜脊椎动物中丢失。Gig1和Gig2的同源基因组成了两个新的基因家族，命名为Gig1基因家族和Gig2基因家族。进化和表达特征分析揭示这基因家族的不同基因拷贝发生了功能歧化。本项目的研究结果有助于理解鱼类具有自身特点的干扰素抗病毒反应机制。