NV蛋白介导HIRRV逃逸宿主RLRs抗病毒信号的分子机制

Hirame rhabdovirus (HIRRV) is a Novirhabdovirus resulting in lethal disease in wild and farmed Japanese ounder (Paralichthys olivaceus) with a severe economic losses in aquaculture industry. So far, the mechanism for HIRRV infection and pathogenesis was remained obscure. Especially, the production and development of an effective strategy against HIRRV infection was hampered due to a limited knowledge on HIRRV-host interaction. Previously, we have found that HIRRV infection could block host cell production in infected cells, and nonstructural protein NV might function as an IFN antagonist. By using molecular techniques such as NV functional complementation analysis, reverse genetics technology, real-time RT-PCR, GFP reporting system, and NV protein-RNA interactions, we will further investigate into the molecular mechanisms of HIRRV evasion from RLRs mediating recognition, signalling and gene expression. Two key questions are to be answerd: firstly, is the evasion of HIRRV achieved by avoidance of sensing by PLRs, blocking of the action of PLRs, and inhibition of signaling pathways and gene expression? Secondly, does the nonstructural protein NV contribute to HIRRV evasion from RLRs signaling? With the assays designed as above, our study will shed light on the molecular mechanism of HIRRV evasion which will be valuable for engineering a promising live HIRRV vaccine strain.

牙鲆是我国海水养殖的重要种类。牙鲆弹状病毒是近来发现的对牙鲆高度致死的病原，严重影响了我国牙鲆养殖产业。一直以来，人们对该病的感染与致病机理等基础科学问题知之甚少，直接制约着相关防控技术和产品的研制与开发。申请人在开展HIRRV研究过程中，偶然发现了该病毒抑制IFN信号通路的现象，暗示IFN上游信号通路即RLRs信号通路可能受到HIRRV NV蛋白阻断。为此，本研究将在前期重要发现的基础上，通过NV功能互补分析、反向遗传NV缺陷HIRRV重组病毒构建、GFP报告系统、NV蛋白-RNA相互作用、NV蛋白-RLRs组件相互作用等分子病毒学方法，围绕HIRRV是否阻断RLRs信号通路，哪个作用环节，何种作用方式，HIRRV NV是否介导了RLRs阻断效应等四个问题展开深入研究，以期揭示NV蛋白介导HIRRV逃逸宿主RLRs抗病毒信号的分子机制，从而为阐明HIRRV感染和致病机理提供科学依据。

RLRs信号通路是各种类型细胞广泛表达的细胞质病毒RNA的模式识别通路，介导细胞抗病毒免疫反应。本项目通过对HIRRVNV蛋白功能互补分析，发现牙鲆弹状病毒的NV蛋白可互补重组病毒IHNV-ΔNV-GFP 的RlRs逃逸缺陷；通过反向遗传技术构建了NV 缺失重组HIRRV，接种EPC细胞，发现HIRRVNV蛋白能有效增加NV蛋白缺失重组病毒rIHNV-ΔNV-GFP滴度升高90倍；NV 缺失重组HIRRV人工感染牙鲆鱼苗，Real-timeRT-PCR跟踪宿主RLRs信号通路基因（IFN-I、IRF-3、MDA5）在接毒1-15天的动态表达，结果发现，和野生型HIRRV中国株相比，IFN上游信号（MDA5、IRF3）基因和IFN-I下游信号（即IFN刺激基因呈抑制显著消除；通过RFP、GFP共定位，确定HIRRV NV至少干扰了牙鲆RLRs 信号通路中的LGP2、IFN-I环节；HIRRV NV与病毒RNA的蛋白质-RNA 互作、HIRRV NV 和RLRs体外蛋白互作等分析，初步确认NV蛋白至少通过与病毒RNA互作，介导了HIRRV逃逸牙鲆RLRs信号通路。