鳜弹状病毒N蛋白与鳜c-Myc互作调控谷氨酰胺代谢机制  

Identification of interaction between SCRV-N protein and Sc-c-Myc and its regulation of glutamine metabolism

作　　者：张秋爽 叶彩媚 牛银杰 林强[1] 梁红茹[1] 罗霞[1] 李宁求[1] 付小哲[1] ZHANG Qiushuang;YE Caimei;NIU Yinjie;LIN Qiang;LIANG Hongru;LUO Xia;LI Ningqiu;FU Xiaozhe(Key Laboratory of Fishery Drug Development,Ministry of Agriculture and Rural Affairs,Guangdong Provincial Key Laboratory of Aquatic Animal Immune Technology and Green Breeding,Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380,China;College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]中国水产科学研究院珠江水产研究所,农业农村部渔用药物创制重点实验室,广东省水产动物免疫与绿色养殖重点实验室,广东广州510380 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《水产学报》2024年第5期54-62,共9页Journal of Fisheries of China

基　　金：国家重点研发计划(2023YFD2400701);广州市重点研发计划(2024B03J1263);中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金资助(2023TD48)。

摘　　要：为了研究鳜弹状病毒(SCRV)如何调控鳜c-Myc(Sc-c-Myc)进而调控谷氨酰胺代谢的分子机制,本研究通过免疫共沉淀联合蛋白质谱寻找可能与Sc-c-Myc互作的病毒蛋白,初步分析确定为核衣壳蛋白(N蛋白);Co-IP结果显示,SCRV的N蛋白与Sc-c-Myc存在相互作用。通过PCR扩增获得了带有Flag标签序列的SCRV-N基因的ORF片段,并构建了SCRV-N蛋白过表达载体pcDNA-N-Flag;将pcDNA-N-Flag质粒转染鳜脑组织细胞系(CPB细胞系),荧光共定位结果显示,Sc-c-Myc与SCRV-N在细胞质内存在共定位现象;通过逆转录实时定量PCR(RT-qPCR)和免疫印记(Western blot)检测转染pcDNA-N-Flag的CPB细胞系中Sc-c-Myc及谷氨酰胺代谢通路关键酶(GLS1、GDH和IDH2)的表达变化,发现Sc-c-Myc、GLS1的转录水平和蛋白水平均显著上调。综上表明,SCRV的N蛋白与Sc-c-Myc互作促进Sc-c-Myc的表达,进而调控宿主细胞谷氨酰胺代谢途径,为SCRV防控提供了新的靶点。With the continuous development of Siniperca chuatsi culture technology,the scale of S.chuatsi culture industry in China has been expanding.However,in recent years,outbreaks of S.chuatsi viral diseases have become more frequent,causing serious economic losses to farmers.Among them,S.chuatsi rhabdovirus(SCRV)disease is one of the most common diseases.Viruses are non-cellular organisms that rely on host cell metabolism to complete their replication and proliferation,and viruses induce host cell metabolic reprogramming to rapidly obtain the biomolecules and energy required for viral replication and proliferation,including glycolysis,glutamine metabolism,lipid metabolism,nucleotide biosynthesis and so on.In turn,c-Myc is a key transcription factor that regulates cellular metabolism,and can regulate the expression of glutaminase1(GLS1),the rate-limiting enzyme of the glutaminase pathway,thereby regulating glutamine metabolism.Moreover,viruses can alter c-Myc protein stability or activity by encoding viral proteins that interact directly or indirectly with c-Myc,thereby regulating cellular metabolism.Therefore,in order to study the molecular mechanism of how SCRV regulates Sc-c-Myc and then regulates glutamine metabolism,the viral protein probably interacting with Sc-c-Myc was identified and analyzed by co-immunoprecipitation(Co-IP)and protein mass spectrometry in this study.SDS-PAGE results showed the specific protein bands of 45-65,65-75,and 100-130 ku in SCRV-infected cells,and protein profiling showed that one viral protein was identified in the Sc-c-Myc IP sample,which was the nucleoprotein(N)of the SCRV,and the peptide intensity value of the hit was high.The Co-IP results showed that SCRV-N interacted with Sc-c-Myc.The SCRV-N ORF with Flag tag was obtained by PCR,and the pcDNA-N-Flag plasmid was constructed.Then pcDNAN-Flag plasmids were transfected into Chinese perch brain cells(CPB cells),and fluorescence microscopy observation found that SCRV-N colocalized with Sc-c-Myc in the cytoplasm.Furthermore,quantitativ

关 键 词：鳜 鳜弹状病毒(SCRV) 鳜c-Myc 蛋白互作 谷氨酰胺代谢 

分 类 号：S942[农业科学—水产养殖]