鳜弹状病毒调控谷氨酰胺还原性代谢途径促进自身增殖

Reductive glutamine metabolism promotes the efficient replication of Siniperca chuatsi rhabdovirus in Chinese perch brain cells

为探究鳜弹状病毒(Siniperca chuatsi rhabdovirus,SCRV)复制增殖与谷氨酰胺还原性代谢(reductive glutamine metabolism,RGM)途径的相互作用关系,以鳜脑细胞系(Chinese perch brain cells,CPB)为增殖体系,采用实时荧光定量PCR技术检测病毒拷贝数和RGM途径关键酶mRNA表达水平变化,并通过蛋白免疫印迹方法检测RGM途径关键酶蛋白表达水平变化。结果发现,当培养基缺失谷氨酰胺时SCRV拷贝数降低了99.5%,表明谷氨酰胺是SCRV增殖所必需;而SCRV感染上调CPB细胞RGM途径关键酶的表达,其中异柠檬酸脱氢酶2(isocitrate dehydrogenase 2,IDH2)上调倍数最大,表明SCRV感染上调了细胞RGM途径中关键酶基因的表达;同时,抑制细胞RGM途径关键酶的表达导致SCRV病毒产量显著下降,表明RGM途径有利于SCRV复制增殖;进一步敲降细胞IDH2基因表达可显著抑制SCRV N蛋白表达,而过表达细胞IDH2基因可显著促进SCRV N蛋白表达,表明IDH2在病毒增殖中具有重要作用。综上所述,SCRV感染可调控CPB细胞RGM通路以满足其自身增殖,其结果可为阐明鳜弹状病毒病致病机制和抗病毒治疗策略提供新的思路。

Viruses rely on host cellular metabolism for energy and macromolecule synthesis during replication.Therefore,there is a close relationship between viral replication and cellular metabolism.Siniperca chuatsi rhabdovirus(SCRV)causes significant economic losses in the Chinese perch(Siniperca chuatsi)industry.Thus,it is important to address the interaction between SCRV replication and host cells to prevent and control SCRV.However,little is known about the relationship between SCRV replication and cellular metabolism.In addition,glutamine is an abundant amino acid necessary for energy generation and macromolecule synthesis in cells.Glutamine is also essential for the infection and replication of some viruses.Glutaminolysis and reductive glutamine metabolism(RGM)are the main pathways of glutamine metabolism.Particularly,the RGM pathway plays an important role in tumor cells.However,the metabolic pathway of glutamine involved in viral replication remains largely uncharacterized.To clarify the interaction between SCRV replication and RGM,we analyzed the expression of key enzymes in the RGM pathway in Chinese perch brain(CPB)cells following infection with SCRV,using quantitative real-time PCR(qRT-PCR)and western blotting.Our results showed that SCRV replication was decreased in CPB cells cultured in a glutamine-depleted medium,indicating that glutamine is required for efficient SCRV replication.In addition,SCRV infection promoted the expression of key enzymes involved in the RGM pathway in CPB cells,particularly the expression of isocitrate dehydrogenase 2(IDH2),indicating that RGM in CPB cells is altered during SCRV infection.SCRV proliferation was also inhibited in CPB cells cultured in the presence of bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide(a glutaminase inhibitor),(–)-epigallocatechin monogallate(a glutamate dehydrogenase inhibitor),5-(tetradecyloxy)-2-furoic acid(an acetyl-CoA carboxylase inhibitor),or 4-methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid(a fatty acid synthase inhibitor),suggesting that the RGM pathway is beneficial for SCRV replication and proliferation.Furthermore,downregulation of the IDH2 gene,the product of which convertsα-ketoglutarate(α-KG)to citrate,significantly inhibited the yield of SCRV and the expression of the SCRV structural protein.Conversely,overexpression of the IDH2 gene significantly increased the yield of SCRV and the expression of SCRV structural protein,indicating that IDH2 plays an important role in SCRV replication and proliferation.These results indicated that SCRV infection induces RGM to accommodate the biosynthetic and energy needs required for its efficient replication and proliferation.These findings provide new insights into viral pathogenesis and antiviral treatment strategies.