1 Molecular cloning , Expression and Functional 2 Analysis of Interferon Regulatory Factor 5 ( IRF 5 ) in 3 Malabar grouper ( Epinephelus malabaricus ) 4

Thirunavukkarasu Periyasamy 1, Ming-Wei Lu 1, 2, * 5 1 Laboratory of Molecular Virology and Immunology, Department of Aquaculture, The College of 6 Life Science, National Taiwan Ocean University, No. 2, Beining Road, Keelung 202, Taiwan; 7 [email protected] 8 2 Center of Excellence for the Oceans, National Taiwan Ocean University, No. 2, Beining Road, 9 Keelung 202, Taiwan; [email protected], [email protected] 10 * Correspondence: [email protected]; Tel.: +886-24622192-5206 11 12 13 14 15 Abstract: Interferon regulatory factor 5 (IRF5) is known to be involved in the innate immune 16 response and pro-inflammatory cytokines. However, the roles of IRF5 in immune responses in 17 Malabar grouper (Epinephelus malabaricus) have not been extensively explored. In this study, IRF5 18 gene was identified and characterized from M. grouper. The full-length IRF5 cDNA consisted of a 5’ 19 terminal untranslated region (5’-UTR) of 289 bp and a 3’-UTR of 542 bp, an open reading frame 20 (ORF) of 1500 bp encoding a polypeptide of 499 amino acids with a predicted molecular mass of 21 56.28 kDa and isoelectric point (pI) of 5.2. The putative MgIRF5 protein consists of four important 22 conserved domains: a helix DNA-binding domain (DBD) at the N-terminus, a middle region, an IRF 23 association domain (IAD) and a virus activated domain (VAD) at the C-terminus. Sequence 24 alignment and phylogenetic analysis showed that highest sequence similarity of IRF5 was observed 25 between the IRF5 genes from Oplegnathus fasciatus and Miichthys miiuy. The mRNA transcripts of 26 IRF5 were detected in a wide range of tissues types from healthy M. grouper with highest 27 expression in muscle, liver and skin. After treatment with poly (I: C), it was significantly 28 up-regulated in spleen and liver tissues. When infected with NNV, the expression level of MgIRF5 29 was up-regulated in spleen and head kidney and their transcriptional responses to IRF5 increased 30 in the grouper kidney cells. This approach suggests that MgIRF5 is important in the underlying 31 mechanism of the innate immune responses against antiviral response. 32 33