| IRF8(Interferon regulatory factors 8)and IRF2 are members of the interferon regulatory factor family.IRF8 includes a highly conserved N-terminal DNA binding domain(DBD)and a less conserved C-terminal IRF-associated domain(IAD).IRF8 plays an important role in cell growth and differentiation,tumorigenesis and innate immunity.IRF2 contains three domains:N-terminal DBD region,C-terminal IAD2 region and TRD region.IRF2 can also regulate cell development and differentiation and is induced by interferon,which plays an important role in innate immunity.In the transcriptional regulation of virus-stimulated immune genes,IRF8 interacts with IRF2 to form a complex.The complex regulates the expression of downstream genes by competing with IRF1 for the binding site on the ISRE element.In order to study the role of grass carp IRF8(CiIRF8)and IRF2(CiIRF2)in the regulation of gene expression,we analyzed the tissue expression levels of CiIRF8 and CiIRP2 by Q-PCR.The results showed that CiIRF8 and CiIRF2 are expressed in grass carp brain,eyes,intestines,gills,skin,liver,spleen,and kidney tissues.After Poly I:C stimulation,the expression levels of CiIRF8 and CiIRF2 in each tissue were up-regulated.However,the response time,degree of CiIRF8 and C/IRF2 are different.In detail,the expression level of CiIRF8 reached the highest in seven tissues of brain,eye,intestine,skin,liver,spleen,and kidney at 6 h,but in gill tissue at 12 h.And the expression level of CiIRF8 is the highest in spleen tissue after stimulation.The expression level of CiIRF2 in all tissues reached the highest at 6 h,the expression level of CiIRF2 is the highest in liver tissue.Thses results proved that CiIRF8 and CiIRF2 may have different effects in various tissues to respond the virus.In additon,the results also anlyzed by Q-PCR showed that the expression levels of CiIRF2 and CiIRF8 in grass carp CIK cells both increased significantly after Poly I:C stimulation.In order to study the role of IRF8 in antiviral immunity of grass carp,we analyzed the expression of CiIOFN on mRNA and protein level by Q-PCR and Western blot in CIK cells after overexpression of CiIRF8,respectively.The results showed that the expression levels of CiIFN were reduced on mRNA and protein level in CIK cells overexpressing CiIRF8.On the contrary,after knocking down CiIRF8,the expression of CiIFN was up-regulated.Therefore,we may conclude that CiIRF8 can negatively regulate the expression of CiIFN.Then we studied the location distribution of CiIRF2 and CiIRF8 in the CIK cells by confocal fluorescence microscopy.The results showed that CiIRF2 was distributed in the nucleus,and CiIRF8 was evenly distributed in cells.After Poly I:C stimulation,there was no change in the distribution of CiIRF2,while CiIRF8 had translocated from cytoplasm to nucleus.In order to explore the existence of CiIRF8/CiIRF2 complex,we co-transfected the recombinant plasmids of FLAG-tagged CiIRF2 and GFP-tagged CiIRF8 into HEK-293T cells.Immunoprecipitation detection revealed that CiIRF8 can interact with CiIRF2.In addition,immunofluorescence co-localization results also showed that CiIRF8 interacts CiIRF2 in the nucleus.To further study the role of CiIRF8/CiIRF2 complex in antiviral immunity of grass carp,we transfected the plasmids with pcDNA3.1-tagged CiIRF8,pcDNA3.1-tagged CiIRF2 and co-transfected pcDNA3.1-tagged CiIRF8 and pcDNA3.1-tagged CiIRF2 plasmid into CIK cells,respectively.Double luciferase experiments showed that CiIRF8 and CiIRF2 negatively regulate the transcription activity of CiIFN promoter,and the negative regulation of CiIRF8/CiIRF2 complex on CiIFN transcription activity is more significant.This result indicates that CiIRF8 enhances the negative regulation of CiIFN transcription by interacting with CiIRF2. |
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