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

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,quantitative reverse transcription PCR(RT-qPCR)and Western blot were used to detect the expression changes of Sc-c-Myc and key enzymes in the glutamine metabolism pathway(GLS1,GDH,and IDH2)in CPB cells transfected with pcDNA-NFlag.RT-qPCR results showed that compared with the control,the expression of Sc-c-Myc and GLS1 mRNA was significantly upregulated after transfection with SCRV-N,increasing by 2.1 and 3.1 times,respectively,while the expression changes of GDH and IDH2 mRNA were not significant.Western blot results showed that the expression of Sc-c-Myc and GLS1 protein was significantly increased after transfection with SCRV-N,while the expression changes of GDH and IDH2 were not significant.In summary,it is observed that SCRV interacts with Sc-c-Myc through the N protein,thereby regulating the glutamine metabolism pathway to meet its own replication and proliferation needs.This study aimed to explore how SCRV regulates glutamine metabolism by interacting with Scc-Myc,investigating the role of Sc-c-Myc in SCRV-induced glutamine metabolism remodeling.The findings reveal that SCRV interacts with the Sc-c-Myc through the N protein,preliminarily uncovering the molecular mechanism of SCRV-induced glutamine metabolism remodeling.Therefore,it provides a theoretical basis and new insights for understanding the pathogenic mechanism of SCRV and for the prevention and treatment of SCRV.