THE INTERFERON INDUCED PROTEIN-35 kDa NEGATIVELY REGULATES RIG-I CASCADE GENES IN ROCK BREAM

N. C. N. Perera, Hanchang Sohn, G. I. Godahewa, and Jehee Lee
 
Department of Marine Life Sciences & Fish Vaccine Research Center
Jeju National University
Jeju Self-Governing Province 63243, Republic of Korea
E-mail: chathunp@gmail.com
 

Viral diseases are a serious threat to both natural and cultured fish populations. They may lead to massive economic losses to the global aquaculture industry. IFNs play a major role in the antiviral defense mechanism of the innate immune response in vertebrates. Interferon induced protein-35 kDa (IFP35) is an antiviral protein induced by Type II IFN (IFN-γ). In this study, we have identified IFP35 from the previously constructed rock bream cDNA database (RbIFP35) and characterized with the online bioinformatics tools. Moreover, physiological features of the RbIFP35 were evaluated through a spatial distribution where the immune related responses were evaluated through an immune challenged experiment. Moreover, the effect of RbIFP35 towards RIG-1 antiviral signaling was observed via evaluating the modulation of selected genes in the RIG-1 antiviral signaling pathway including; MAVS, IRF3 and IRF7. Here, we have transfected RbIFP35 into rock bream heart cell lines, treated with Poly I:C and isolated the cells in several time points. Then we have extracted the total RNA and made the cDNA for the qPCR analysis. The full length cDNA sequence of RbIFP35 contained an ORF of 1113 nucleotides which consists with 371 amino acids. Interestingly, in silico analysis showed that the RbIFP35 sequence contains two characteristic Nmi/IFP 35 domains as previously found in rockfish. Additionally, RbIFP35 shared 70% of identity with Larimichthys crocea and also closely cladded with IFP35 from Larimichthus crocea in the phylogenetic tree. According to spatial distribution a ubiquitous expression was observed among all the selected tissues of healthy rock bream fish but with different magnitudes. The highest expression was observed in the gill tissue, followed by liver and blood. Transcriptional distribution in liver tissue showed that RbIFP35 significantly up-regulated against different stimuli including; Poly I:C, rock bream Irido virus, Edverdisella tarda and Streptococcus iniae. The highest expression of RbIFP35 against Poly I:C, RBIV and S. iniae was observed at 48 h p.i. where the highest expression against LPS was observed at 6 h p.i. However, intense expression against E. tarda was observed at 24 h p.i revealing the modulation of RbIFP35 was differ according to the nature of the pathogenesis. Additionally, cell stimulation assay showed that RbMAVS, RbIRF3 and RbIRF7 were down-regulated by the transfected RbIFP35 in the rock bream heart cells. Collectively, we could conclude that RbIFP35 is an important immune related gene in the rock bream which may important for the innate immunity of the rock bream.