The Roles Of Three Immune-Lipid Metabolism Related Genes Of Grouper In Infection Of SGIV And RGNNV Viruses

As a kind of important economic fish developed in South China and Southeast Asia,grouper(Epinephelus spp.)frequently suffered from the outbreaks of viral disease,which have seriously restricted the healthy and sustainable development of grouper culture and caused heavy economic losses to grouper farming industry.Singapore grouper iridovirus(SGIV)and red spotted grouper nerve necrosis virus(RGNNV)are two important viral pathogens.To date,the pathogenic biology and infection mechanism of SGIV and RGNNV have been completed,while the metabolic changes induced by virus infection and the function of immune-metabolic related gene are still limited.Here,the metabolism alteration induced by RGNNV infection and the functions of interferon induced and lipid metabolism related genes,including cholesterol 25-hydroxylase(CH25H),viperin and interferon-induced transmembrane proteins(IFITMs),were studied.It will not only reveal the roles of fish immunemetabolic related genes in the infection of SGIV,a large DNA virus and RGNNV,a small RNA virus,but also provide more effective new ideas and strategies for the prevention and control of marine fish virus disease.The main innovation research results obtained in this study are listed as follows:1.RGNNV infection significantly changed the metabolic pathway of host cells.The metabolic changes in grouper spleen cells(GS)during RGNNV infection were studied by global metabolic profiling.As infection progressed,71 intracellular metabolites were significantly altered in RGNNV-infected cells compared to mockinfected cells.The levels of metabolites involved in amino acid biosynthesis and metabolism were significantly decreased,while those that correlated with fatty acid synthesis were significantly upregulated during RGNNV infection.Among them,tryptophan and oleic acid were assessed as the most crucial biomarkers for RGNNV infection.In addition,RGNNV infection induced the formation of lipid droplets and relocalization of fatty acid synthase(FASN),indicating that RGNNV induced and required lipogenesis for viral replication.The exogenous addition of palmitic acid(PA)enhanced RGNNV infection,and the inhibition of FASN and acetyl-Co A carboxylase(ACC)significantly decreased RGNNV replication.Additionally,not only inhibition of palmitoylation or phospholipid synthesis,but also destruction of fatty acid β-oxidation significantly decreased viral replication.These data suggested that cellular fatty acid synthesis,including mitochondrial β-oxidation was essential for RGNNV to complete the viral life cycle.2.Ec CH25 H inhibited the entry and replication of SGIV and RGNNV virus by positively regulating interferon response and inflammatory response.We cloned and characterized a CH25 H homolog from orange-spotted grouper(E.coioides)(Ec CH25H).Ec CH25 H encoded a 271-amino-acid polypeptide,with 86% and 59%homology with yellow croaker(Larimichthys crocea)and humans(Homo sapiens),respectively.Ec CH25 H contained a conserved fatty acid(FA)hydroxylase domain and an ERG3 domain.Ec CH25 H was distributed in all detected tissues of grouper,and was induced by RGNNV or SGIV infection,lipopolysaccharide(LPS)or poly(I:C)treatment in vitro.Subcellular localization analysis showed that Ec CH25 H and mutant Ec CH25H-M were distributed in the cytoplasm and partly colocalized with the endoplasmic reticulum(ER).In vitro,Ec CH25 H overexpression or pretreatment with25-hydroxycholesterol(25HC)significantly inhibited the entry and replication of SGIV and RGNNV,and its antiviral activity was related to enzyme activity.On the contrary,knockdown of Ec CH25 H significantly promoted virus entry and replication.Besides,Ec CH25 H overexpression positively regulated the interferon(IFN)-related molecules and proinflammatory cytokines,and enhanced IFN-1,IFN-3 and interferon stimulation response element(ISRE)promoter activity.Furthermore,Ec CH25 H overexpression reduced the total cholesterol content,while knockdown of Ec CH25 H increased the total cholesterol content in GS cells.3.Ec IFITMs affected virus infection through regulating lipid metabolism.The IFITM homologues from E.coioides were cloned and identified,which were named as Ec IFITM1 and Ec IFITM3,respectively.Ec IFITM1 and Ec IFITM3 encoded a 131-amino-acid polypeptide and a 148-amino-acid polypeptide,respectively,which both contained five domains: NTD,IMD,CIL,TMD and CTD.Ec IFITM1 and Ec IFITM3 were differently distributed in all detected tissues of grouper.Besides,SGIV or RGNNV infection,LPS or poly(I:C)treatment,significantly upregulated the transcription level of Ec IFITM1 and Ec IFITM3.Ec IFITM1 was mainly distributed in the cytoplasm,and accumulated around the nucleus in dots,which was colocalized with early endosomes(EE)and lysosomes in varying degrees,while Ec IFITM3 was mainly distributed in the cell membrane and in little cytoplasmic dot-like aggregations.In vitro,overexpression of Ec IFITM1 and Ec IFITM3 significantly inhibited the entry and replication of SGIV and RGNNV,while knockdown of Ec IFITM1 and Ec IFITM3 significantly promoted the entry and replication of virus.In addition,Ec IFITM1 did not affect the content of cholesterol in the cells,but was colocalized with the lipid droplets,and significantly regulated cells lipid metabolism,while Ec IFITM3 overexpression increased the content of total cholesterol in GS cells.4.Ecviperin inhibited SGIV replication by positively regulating interferon and inflammation response.A viperin homolog from orange spotted grouper(E.coioides)(Ecviperin)was cloned and its antiviral function was clarified in this study.Ecviperin encoded a 361-aa protein which shared 87% and 69% identity with Siniperca undulata and Homo sapiens,respectively.Amino acid alignment analysis showed that Ecviperin contained a conserved radical-SAM domain(aa 73-281).In healthy grouper,Ecviperin was distributed in all detected grouper tissues,and the expression of Ecviperin was the highest in kidney and spleen.In vitro,the m RNA expression of Ecviperin was significantly upregulated in response to SGIV infection.Ecviperin was distributed in the cytoplasm and co-localized with ER.The ectopic expression of Ecviperin significantly inhibited the replication of SGIV.Furthermore,overexpression of Ecviperin positively regulated the expression of IFN related molecules and proinflammation cytokines,and enhanced the activity of IFN-1,ISRE and nuclear factor of kappa B(NF-κB)promoter.Further analysis showed that Ecviperin promoted the interferon response mediated by poly(I:C)and Ec STING,and interacted with Ec STING.