Molecular Cloning And Immune Function Analysis Of IRF In Common Carp (Cyprinus Carpio L.)

Interferon regulatory factor (IRF) genes encode a family of DNA-binding proteins that areinvolved in the transcriptional regulation of type-l Interferon (IFNs) and IFN-stimulated genes(ISGs) and play critical roles in viral response, cytokine signalling, cell growth regulation andhematopoietic differentiation. There are9members of IRF family in mammals, including IRF-1,IRF-2, IRF-3, IRF-4/PIP/LSIRF/ICSAT, IRF-5, IRF-6, IRF-7, IRF-8/ICSBP andIRF-9/ISGF3γ/p48, while11members including2novel IRF named IRF-10and IRF-11, wereidentified in fish. All IRF family members share significant homology in the N-terminal115amino acids (aa), which comprise the DNA binding domain (DBD) and is characterized by fivetryptophan repeats. Except IRF-1and IRF-2, all other IRF family members contain the IRFassociated domain (IAD) which mediates interactions in the3’ terminal of the protein. Based onthe structural and functional similarity, the IRF family members can be divided into foursubfamilies, including IRF-1subfamily, IRF-3subfamily, IRF-4subfamily and IRF-5subfamily.The studies of mammalian IRF family members found that IRF-1, IRF-3, IRF-5and IRF-7areinvolved in the positive-feedback regulation of type ⅠIFN genes and play critical roles in theantiviral innate responses. Recently, the similar IFN system was found in fish as in mammals,and considering the important roles of IRF in the IFN system, the studies of fish IRF familymembers are increasing. Common carp (Cyprinus carpio L.) is one of the most importantfreshwater aquaculture species in China, but this farmed fish is prone to virus infection, soinvestigating its antiviral mechanisms is of great importance. So far, IRF-3and IRF-7have beenidentified in common carp, so in this study we cloned the full-length cDNA of common carpIRF-1and IRF-5, and studied their immune functions by analysing their tissue distributions andexpressions post poly I:C injection.The full-length cDNA of common carp IRF-1was1427nucleotides (nt), consisting of a5’untranslated region (5’-UTR) of126nt, a3’-UTR of440nt, and an open reading frame (ORF) of861nt encoding286amino acids. The IRF-1protein contained a DBD in the N-terminal, sixtryptophan residues were present in the DBD. The full-length cDNA of common carp IRF-5was2042nt, consisting of a5’-UTR of266nt, a3’-UTR of222nt, and an ORF of1554nt encoding517amino acids. The IRF-5protein contained a DBD in the N-terminal and an IAD in the C-terminal, five tryptophan residues were present in the DBD. The identity of common carpIRF-1with other IRF-1proteins varied from43.7%to78.4%, the identity of DBD were71.9%-93.0%, the identity of C-terminal were only13.8%-69.5%. Common carp IRF-1showedthe highest whole identity to zebrafish IRF-1and the DBD was most similar to grass carp IRF-1.The identity of common carp IRF-5with other IRF-5proteins varied from50.3%to90.0%, theidentity of DBD were58.8%-96.5%, the identity of IAD were63.0%-97.3%. Common carpIRF-5showed the highest whole identity to grass carp IRF-1, with the highest identity of DBDand IAD. The phylogenetic analysis showed that common carp IRF-1and IRF-5belonged toIRF-1subfamily and IRF-5subfamily respectively, they had close relationship with theirmammalian and other fish counterparts. The analysis above indicates common carp IRF-1andIRF-5were conserved among vertebrates.Real-time PCR results showed common carp IRF-1and IRF-5genes were constitutivelyexpressed in all the tissues examined, although the expression levels were different. Commoncarp IRF-1was found to be most highly expressed in spleen, while highest expression level ofIRF-5was in gill, the general expression level of IRF-1was higher than that of IRF-5. In order tostudy the roles of common carp IRF-1and IRF-5in the antiviral immune responses, commoncarps were injected intraperitoneally with poly I:C solution, then the expression levels of sevenimmune-related organs (head kidney, liver, spleen, gill, foregut, hindgut and skin) wereexamined at3h,6h,12h,1d,2d,3d and5d post injection. The expression patterns ofcommon carp IRF-1were similar in the seven organs, the expression of carp IRF-1increasedimmediately after injection, reached peak level at a early time, then gradually decreased to thebasal level. The fastest and highest induction of IRF-1occurred in the foregut, the expressionlevel increased to22.1-fold after injection. The expression patterns of common carp IRF-5postinjection were different in seven organs, the expression level changed a little in head kidney,liver, hindgut and skin, significant increases were in foregut, spleen and gill, they increased to22.8-fold，4.1-fold and4.2-fold respectively. All the results show that common carp IRF-1andIRF-5may play important roles in the antiviral innate responses, and the activation of IFN byIRF-5may be virus-specific and cell type-specific as mammalian and other fish IRF-5proteins,the antiviral mechanism of IRF-5in common carp need further investigation.