Mechanisms Of Reductive Glutamine Metabolism In Multiplication Of Infectious Spleen And Kidney Necrosis Virus And Siniperca Chuatsi Rhabdovirus

Infectious spleen and kidney necrosis virus（ISKNV）and Siniperca chuatsi rhabdovirus（SCRV）are the two most important viruses endangering Siniperca chuatsi aquaculture..The study for their pathogenesis may help to establish more effective strategies for preventing and controlling ISKNV and SCRV diseases.Viruses are non-cellular micro-organism,they finish their life cycles depending on hosts metabolism.Therefore,cells metabolism reprogramming induced by viruses infection is one of the most important manifestations of interaction of virus and hosts.Both ISKNV and SCRV belong to envelope viruses,and glutamine metabolism can provide precursors for lipid synthesis.Glutamine metabolism mainly includes glutaminolysis pathway and reductive glutamine metabolic（RGM）pathway.Previous studies have shown that ISKNV multiplication depend on glutamine and lead to its metabolic programming,but it is not clear which metabolic pathway of glutamine plays a role in the multiplication of virus.Therefore,it is very meaningful to explore the role of glutamine metabolism in virus multiplication and its mechanism for the development of prevent and controll virus diseases.In this study,we aimed to address the scientific issue that how ISKNV and SCRV induce CPB cells glutamine metabolism reprogramming.Using Chinese perch brain cells（CPB）as a model,we explored the the regulatory mechanism of CPB cells infected with ISKNV or SCRV on RGM to provide a new sight for elucidate the pathogenic mechanism of ISKNV and SCRV.The main results are as follows.1.The effect of ISKNV infection on reductive glutamine metabolic pathwayCombined with transcription,proteome and metabolic analysis,the results showed that changes in the RGM pathways in ISKNV-infected CPB cells at 24 and 72 hpi are shown in Fig 1-3.At 72 hpi,but not at 24 hpi,there was significant up-regulation of related enzymes of RGM—Glutaminase（GLS）,Glutamate dehydrogenase（GDH）,Isocitrate dehydrogenase 1（IDH1）,Isocitrate dehydrogenase 2（IDH2）,ATP-citrate lyase（ACLY）,suggesting that ISKNV infection enhanced RGM mainly at the late stage of ISKNV replication cycle.In addition,the results of real-time q PCR showed that ISKNV promote transcription of GLS,GDH,IDH1 and IDH2.And the results of western blot showed that ISKNV promote protein expression of GLS,GDH and IDH2 and ACLY.In conclusion,CPB cells infected with ISKNV could induce changes in RGM pathway,especially the expression of IDH2 in RGM pathway.The regulation of ACLY might focus on the stage of virus release.Based on finding ISKNV infection could promote RGM of CPB cells,we utilized inhibitors to regulate RGM to explore the effect of reductive glutamine metabolic on ISKNV multiplication.The results of real-time q PCR showed that the bis-2-（5-phenylacetamido-1,3,4-thiadiazol-2-yl）ethyl sulfide（BPTES）,5-（tetradecyloxy）-2-furoicacid（TOFA）and4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid（C75）has no significant effect on ISKNV during replication stage（36hpi）,but could inhibit ISKNV multiplication during release stage（72hpi）.In addition,the results of real-time q PCR and western blot showed that inhibitors can inhibit the expression of structure protein of ISKNV.The results showed that the four inhibitors could significantly inhibit ISKNV structural protein expression in m RNA level and MCP protein level.In conclusion,ISKNV regulates the RGM pathway to facilitate the synthesis of viral envelope lipids.2.The effect of SCRV infection on reductive glutamine metabolic pathwayFu etc^[1]showed that Glutamine was not essential for the viability of CPB cells,but it was required for efficient ISKNV multiplication.In addition,the anaplerosis efficiency of citric acid was significantly higher than those ofα-KG,OAA,and pyruvate.In this study,we have investigated the relationship between glutamine and SCRV multiplication,the results showed that Glutamine was also required for efficient SCRV multiplication,indicating that SCRV multiplication also require glutamine to provide it with biomolecules and energy.Furthermore,the results of real-time q PCR showed that SCRV promote transcription of GLS,GDH,IDH1,IDH2 and ACLY.And the results of western blot showed that SCRV promote protein expression of GLS,GDH,IDH2 and ACLY.In conclusion,CPB cells infected with SCRV also could induce changes in RGM pathway,especially the expression of IDH2 in RGM pathway.Based on finding SCRV infection could promote RGM of CPB cells,we also utilized inhibitors to regulate RGM to explore the effect of reductive glutamine metabolism on SCRV multiplication.The results showed that BPTES and TOFA significantly inhibited the yield of SCRV during replication stage（4hpi）,and BPTES,（–）-epigallocatechinmo nogallate（EGCG）and TOFA significantly inhibited the yield of SCRV during release stage（12hpi）.In addition,the results of real-time q PCR and western blot showed that four inhibitors can inhibit the expression of structure protein of SCRV.In conclusion,SCRV also regulates the RGM pathway to facilitate the synthesis of viral envelope lipids.3.The effect of IDH2 on virus multiplicationThe relationship between the expression of IDH2 gene and ISKNV and SCRV multiplication was investigated by constructing IDH2knockdown cell line and overexpression cell line.The results showed that overexpression of IDH2 gene promotes ISKNV and SCRV virus production,and facilitates the expression of viral structural proteins.Studies have shown that RGM in cancer cells mediated by the ratio of NAD（P）⁺/NAD（P）H.In order to detect whether the effect of ISKNV and SCRV infection on RGM in CPB cells was mediated by NAD⁺/NADH.So,we detected the ratio of NAD⁺/NADH,the results showed that the rate of NAD⁺/NADH increased,when CPB cells were infected with ISKNV and SCRV respectively.And IDH2 gene enhance the ratio of NAD⁺/NADH.In summary,ISKNV and SCRV infection regulate the increase the ratio of NAD⁺/NADH,and then promote the expression of IDH2,and then promote the formation of lipids in RGM pathway,which is beneficial to virus assembly.