草鱼呼肠孤病毒纤维蛋白VP56与草鱼GRP78蛋白互作诱导内质网应激  

Grass carp reovirus(GCRV)fibrin VP56 interacts with grass carp(Ctenopharyngodon idella)GRP78 for inducting endoplasmic reticulum stress

作　　者：苏航 苏建国 SU Hang;SU Jianguo(College of Fisheries,Huazhong Agricultural University,Wuhan 430070,China;Laboratory for Marine Biology and Biotechnology,Pilot National Laboratory for Marine Science and Technology,Qingdao 266237,China)

机构地区：[1]华中农业大学水产学院,湖北武汉430070 [2]青岛海洋科学与技术国家实验室,海洋生物与生物技术实验室,山东青岛266237

出　　处：《水产学报》2023年第9期155-162,共8页Journal of Fisheries of China

基　　金：国家重点研发计划“蓝色粮仓科技创新”专项(2018YFD0900504)。

摘　　要：为了阐明草鱼呼肠孤病毒(GCRV)的感染机制,深入探究VP56(Ⅱ型和Ⅲ型GCRV特有的纤维蛋白)与宿主细胞蛋白的相互作用。实验通过免疫共沉淀(co-IP)发现,VP56与细胞内定位于内质网的分子伴侣蛋白葡萄糖调节蛋白78(GRP78)发生相互作用。而后,VP56与GRP78的互作通过基于远红外红色荧光蛋白mNeptune的双分子荧光互补系统(BiFC)得到验证。在VP56稳定表达的草鱼肾细胞系(CIK)中,相较于CIK细胞,内质网则形态发生巨大变化,产生肿胀、扩张、脱粒等现象,说明VP56激活内质网应激。通过对内质网应激下游转录因子的mRNA水平检测,发现VP56激活活化转录因子6(ATF6)介导的信号转导途径,激活非折叠蛋白反应。研究表明,GCRV-Ⅱ感染过程中破坏了细胞稳态、激活内质网应激。本研究为GCRV感染机制和抗GCRV病毒研究提供了新思路,揭示了一种新的病毒逃逸机制,有助于淡水养殖产业中草鱼出血病的防控。Grass carp(Ctenopharyngodon idella)is one of the most important aquaculture fish species in China.C.idella hemorrhagic disease caused by grass carp reovirus(GCRV)results in severe outbreaks annually.It causes great losses to aquaculture industry and a threat to food safety.GCRV is the most virulent in aquareovirus.VP56 is a fibrin which is specific to typeⅡ/Ⅲ.It attaches the surface of host cells during GCRV infection.In order to clarify the infection mechanism of GCRV,this study delved into the interaction between VP56 and host cell proteins.First,co-immunoprecipitation(co-IP)was performed.The specific antibodies to different tags respectively fused on the two molecules were used.Empty vectors and negative immunoglobulin G were used as control.It was found that VP56 interacts with the molecular chaperone protein glucose regulated protein 78 ku(GRP78)located in the endoplasmic reticulum.Then,the interaction between VP56 and GRP78 was verified by the bimolecular fluorescence complementation(BiFC)based on the far-red fluorescent protein mNeptune.According to the BiFC results,red fluorescence of mNeptune was observed in CIK cells transfected with both VP56 and GRP78 respectively linked with each terminal of the mNeptune protein,showing that these two fusion proteins presented closely and sent out red fluorescence signal.In VP56 stably expressed C.idella kidney(CIK)cells,the endoplasmic reticulum morphology underwent tremendous changes,compared with empty vector stably expressed CIK cells resulting in swelling,expansion,and degranulation,indicating that VP56 activates endoplasmic reticulum stress.mRNA levels of transcription factors downstream of endoplasmic reticulum stress were detected,which revealed that VP56 activates the signal transduction pathway regulated by activating transcription factor 6(ATF6)and triggers the unfolded protein response.The above results indicate that during GCRV-Ⅱinfection,cell homeostasis is destroyed,and endoplasmic reticulum stress is activated.This study provides new ideas for t

关 键 词：草鱼 草鱼呼肠孤病毒 纤维蛋白VP56 葡萄糖调节蛋白78 内质网应激 

分 类 号：S941.41[农业科学—水产养殖]