The Regulatory Function Of Interferon Regulatory Factors(CgIRF-1 And CgIRF-8)on The Interferon System In Oyster,Crassostrea Gigas

Interferon regulatory factor(IRFs)family is a class of transcriptional regulatory factors with multiple biological functions,which play important roles in regulating the antiviral immune response,immune cell growth and differentiation,and oncogenesis.By far,the detailed regulation mechanism of IRFs on IFN system have been well studied in vertebrates,however,the knowledge in marine invertebrate is still in infancy.In the present study,two IRFs,named CgIRF-1 and CgIRF-8,were identified from Pacific oyster Crassostrea gigas,and their functions in regulating IFN-mediated antiviral immune response were conducted using the methods of molecular biology,cell biology,bioinformatics,and so on.The full length of open reading frames of CgIRF-1 and CgIRF-8 genes were 990 bp and 1425 bp,respectively,which encoded a polypeptide of 229 and 473 amino acids,respectively.The Nterminus of CgIRF-1 and CgIRF-8 were conserved and both contained a characteristic DBD.There was a typical IAD1 domain in the C-terminus of CgIRF-8,which not for CgIRF-1.It was worth noting that,there were some tyrosine,serine and threonine residues in the last 100 amino acid residues of CgIRF-1,whose phosphorylation could enhance the transcriptional activity of IRF-1.The mRNA expression of CgIRF-1 and CgIRF-8 could be examined in all tested tissues,including muscle,gill,gonad,mantle,hepatopancreas and hemocyte,and the highest expression levels was both observed hemocyte.The immunofluorescence analysis showed that the CgIRF-1 protein was located in both the nucleus and cytoplasm of oyster hemocytes,while the CgIRF-8 was mainly distributed in the cytoplasm of oyster hemocytes.The expression level of CgIRF-1 mRNA in hemocytes was significantly up-regulated at 48 h after poly(I: C)stimulation(p < 0.05).The recombinant CgIRF-1(rCgIRF-1)could in vitro interact with classical IFN-stimulated response elements(ISRE).The dual-luciferase reporter assays showed that CgIRF-1 could significantly enhance the activity of interferon-like protein promoter(p < 0.05),while CgIRF-8 could significantly inhibit the activity of interferon-like protein promoter.Further co-transfection assay showed that CgIRF-8 and CgIRF-1 could significantly inhibit the activity of interferon-like protein promoter(p < 0.01).In conclusion,these results indicated that CgIRF-1 and Cg IRF-8 were highly conserved in protein architecture.In addition,CgIRF-1 could response to poly(I: C)stimulation and regulate antiviral immune response by activating IFN system.CgIRF-8 and CgIRF-1 collaborate to inhibit the IFN system to prevent the body from an excessive immune response.These studies laid an important theoretical foundation for further studies on the IRFs of C.gigas,and also provided a reference for studying the antiviral immune regulation mechanism of invertebrate interferon system.