CHARACTERIZATION OF IκB POST-MODIFICATION DURING NODAVIRUS INFECTION IN ORANGE-SPOTTED GROUPER Epinephelus coioides

Chi-Yu Hsieh*, Yu-Lin Tsai, Young-Mao Chen, and Tzong-Yueh Chen
 
Department of Biotechnology and Bioindustry Sciences, Institute of Biotechnology,
Translational Center for Marine Biotechnlogy, and Agriculture Biotechnology Reasearch
Center, National Cheng Kung University, Tainan 70101, Taiwan
cheetaholic1007@hotmail.com

Orange-spotted grouper is one of the important aquaculture fish in Asia, but the industry faces serious losses of groupers dying of nervous necrosis virus (NNV) infection. NNV infection may induce the post-translational modification of inhibitor of kappa B and then activate the transcription factor, NF-κB, which can transfer into nucleus to bind to the DNA sequence, and regulate pro-inflammatory cytokine gene, like TNFα, to modulate immune responses and defense pathogens.

In this study, we isolated the full-length cDNA of the orange-spotted grouper p65 and IκBα named osgp65 and osgIκBα. Both gene and protein levels of osgp65, osgp50 and osgIκB followed by the NNV copy numbers are elevated, which is most significant in brain tissue compared with healthy groupers. Moreover, we transfected GF-1 cell with osgp65 and observed that osgp65 translocated into nucleus in NNV infection. To investigate the function and activity of osgp65 to TNFα promoter, we cotransfected osgp65 and TNFα promoter-Luc or TNFα promoter ΔNFκB-Luc into GF-1 cell, the over-expression of osgp65 stimulated NF-κB-dependent reporter (κB-luc) activity but deletion mutant. In addition, we also infected with NNV and discovered that NNV activated TNFα promoter more than TNFα promoter ΔNFκB-Luc. Together, these results describe that osgp65 and osgIκBα are evolutionarily conserved in orange-spotted grouper with mammals and play important roles in regulating TNFα promoter in NNV infection. These may provide clues to the detailed molecular mechanism underlying immune response regulation in groupers.