More attention has been paid to fish interferon system since it plays a key role in fish innate immune system. A cDNA library of CAB cells has been established for isolation and identification of cellular genes involved in IFN antiviral response. In this study, we cloned and identified the gene NEDD8 Ultimate Buster-1 (NUB1) and its expression was subsequently characterized. The other two genes involved in fish interferon system, interferon regulator factor 3 (IRF3) and interferon regulator factor 7 (IRF7), were characterized on subcellular localization.
NUB1（NEDD8 Ultimate Buster-1）is identified recently as an interferon inducible gene, and overexpression of human NUB1 results in inhibition of cell growth. In the present study, Carassius auratus NUB1 (CaNUB1) gene was cloned from UV-inactivated grass carp haemorragic virus (GCHV)-infected and interferon-producing Carassius auratus blastulae embryonic cells (CAB) by RACE-PCR. The full-length cDNA of CaNUB1 is 2298 bp, which encodes a protein of 589 amino acids. Structurally, the putative CaNUB1 protein contains a ubiquitin like (UBL) motif in N terminus and two ubiquitin associated domains (UBA) in C terminus, showing 41-45% homology to known counterparts from mammals, birds and amphibian. Expression analysis showed that Poly I:C and LPS were able to upregulate the expression of CaNUB1, indicating that CaNUB1 play some roles in fish antiviral immune response.
IRF3 and IRF7 are two transcription factors important for regulation of expression of type I interferon genes and interferon stimulated genes (ISGs). In the present study, Carassius auratus IRF3 (CaIRF3) gene was cloned by homology cloning method. The full-length cDNA of CaIRF3 is 1839bp encoding a 458-amino-acid protein. CaIRF3 protein shows 28-32% homology to known counterparts from mammals and amphibian. Stucturally, CaIRF3 protein possesses a putative DNA-binding domain (DBD) containing five conserved tryptophan repeats, and an IRF association domain (IAD) in the C terminus. Similar to mammalian IRF3 proteins, CaIRF3 has a serine-rich stretch in C terminus, which might be the phosphorylation site. The sequence （70NGDHSVWKRNFRSALRAKGFKM92） is rich in K / R, which may represent a nuclear localization signal (NLS). Subcellular localization analysis revealed that CaIRF3 and CaIRF7 are located in cytoplasm when IRF3 and IRF7 constructs are transfected into COS7 cells, respectively. Transfection of IRF3 and IRF7 constructs followed Poly I:C treatment for 19h showed that a small amount of IRF3 and IRF7 was present in nucleus.