Grouper viperin acts as a crucial antiviral molecule against iridovirus

Virus inhibitory protein, endoplasmic reticulum-associated, IFN-inducible (viperin), is an antiviral protein, induced by interferon (IFN), poly(I:C) and viral infection to exert antiviral function. To investigate the roles of viperin during fish virus infection, a viperin homolog from orange spotted grouper (Epinephelus coioides) (Ecviperin) was cloned and characterized in this study. Ecviperin encoded a 361-aa protein which shared 87% and 69% identity with Siniperca undulata and Homo sapiens, respectively. Amino acid alignment analysis showed that Ecviperin contained a conserved radical-SAM domain (aa73-281). Phylogenetic analysis indicated that Ecviperin showed the nearest relationship with S. undulata. In healthy grouper, Ecviperin was distributed in all tissues, and the expression of Ecviperin was the highest in kidney and spleen. In vitro, the mRNA expression of Ecviperin was significantly up-regulated in response to Singaporean grouper iridovirus (SGIV) infection. Subcellular localization analysis showed that Ecviperin was distributed in the cytoplasm and co-localized with endoplasmic reticulum (ER). The ectopic expression of Ecviperin significantly inhibited the replication of SGIV. Furthermore, overexpression of Ecviperin positively regulated the interferon related molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon stimulated gene 15 (ISG15), myxovirus resistance gene I (MXI), interferon-induced 35-kDa protein (IFP35), and TNF receptor-associated factor 6 (TRAF6). In addition, the expression of pro-inflammation cytokines was differently regulated by Ecviperin overexpression. Furthermore, reporter gene analysis showed that the overexpression of Ecviperin enhanced the activity of nuclear factor of kappa B (NF-κB), IFN-1 and interferon-stimulated response element (ISRE) promoter, suggesting that Ecviperin might restrict SGIV replication by the positive regulation of interferon and inflammatory response. Taken together, our results demonstrated that Ecviperin encoded an ER-localized protein, and exerted antiviral function against fish DNA virus by up-regulating interferon and pro-inflammatory response.