Cynoglossus semilaevis ISG15: A Secreted Cytokine-Like Protein That Stimulates Antiviral Immune Response in a LRGG Motif-Dependent Manner Wei Wang1,2, Min Zhang1, Zhi-zhong Xiao1, Li Sun 1 * 1 Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China, 2 Graduate University of the Chinese Academy of Sciences, Beijing, China Abstract ISG15 is an ubiquitin-like protein that is induced rapidly by interferon stimulation. Like ubiquitin, ISG15 forms covalent conjugates with its target proteins in a process called ISGylation, which in mammals is known to play a role in antiviral immunity. In contrast to mammalian ISG15, the function of teleost ISG15 is unclear. In this study, we identified and analyzed the function of an ISG15 homologue, CsISG15, from tongue sole (Cynoglossus semilaevis). CsISG15 is composed of 162 residues and possesses two tandem ubiquitin-like domains and the highly conserved LRGG motif found in all known ISG15. Expression of CsISG15 occurred in a wide range of tissues and was upregulated in kidney and spleen by viral and bacterial infection. In vitro study with primary head kidney (HK) lymphocytes showed that megalocytivirus infection caused induction of CsISG15 expression and extracellular release of CsISG15 protein. Purified recombinant CsISG15 (rCsISG15) activated HK macrophages and enhanced the expression of immune genes in HK lymphocytes, both these effects, however, were significantly reduced when the conserved LRGG sequence was mutated to LAAG. Further study showed that the presence of rCsISG15 during megalocytivirus infection of HK lymphocytes reduced intracellular viral load, whereas antibody blocking of CsISG15 enhanced viral infection. Likewise, interference with CsISG15 expression by RNAi promoted viral infection. Taken together, these results indicate that CsISG15, a teleost ISG15, promotes antiviral immune response and that, unlike mammalian ISG15, CsISG15 exerts its immunoregulatory effect in the form of an unconjugated extracellular cytokine. In addition, these results also suggest a role for the LRGG motif other than that in protein conjugation. Citation: Wang W, Zhang M, Xiao Z-z, Sun L (2012) Cynoglossus semilaevis ISG15: A Secreted Cytokine-Like Protein That Stimulates Antiviral Immune Response in a LRGG Motif-Dependent Manner. PLoS ONE 7(9): e44884. doi:10.1371/journal.pone.0044884 Editor: Hidde L. Ploegh, Whitehead Institute, United States of America Received February 17, 2012; Accepted August 15, 2012; Published September 18, 2012 Copyright: 2012 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the grants from the National Basic Research Program of China (2012CB114406), the National Natural Science Foundation of China (31025030), and the Knowledge Innovation Program of the Chinese Academy of Sciences (KZCX2-EW-Q213). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lsun@qdio.ac.cn Introduction Interferons (IFNs) play an important role in the innate immunity against viral infection. IFNs bind to their cognate receptors on the target cells and activate the signal transduction pathways involving Jak kinases and the transcription factors of the STAT family [1,2], which in turn activate the transcription of hundreds of IFN-stimulated genes (ISGs) [3,4]. Among the identified ISGs are a group of proteins called ISG15, which are small ubiquitin-like proteins induced rapidly by IFN stimulation [5,6]. ISG15 was first identified in humans as a ,15 kDa protein derived from a 165-residue precursor [7]. Subsequently, ISG15 homologues were discovered in diverse vertebrate species including fish. All ISG15 proteins possess two ubiquitin-like (UBL) domains and a highly conserved C-terminal LRGG sequence, the latter being known as the ubiquitin conjugation motif [8]. In mammals, both intracellular and extracellular ISG15 have been detected. Intracellular ISG15 are conjugated, via the LRGG motif, to target proteins through a process called ISGylation, which resembles largely ubiquitination, the process of formation of ubiquitin conjugates. Ubiquitination involves three enzymes, i.e., ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3). E1 catalyzes adenylylation of the C-terminal di-glycine sequence in the LRGG motif, which is exposed after proteolytic cleavage, while E2 and E3 catalyze transferring of the ubiquitin moiety to the substrate protein [9]. In most cases, the first ubiquitin molecule is attached to the substrate protein through a linkage formed between the C- terminal glycine residue of ubiquitin and a lysine residue of the substrate protein [10]. Poly-ubiquitination is achieved by successive attachment of new ubiquitin molecules to the conjugated ubiquitins. Similar to ubiquitination, ISGylation begins by adenylylation of the C-terminal di-glycine sequence of the LRGG motif, which is followed by successive transfer of ISG15 from E1, E2 and E3 enzymes to the target protein [11]. Unlike ubiquitination, which is known to function in protein and immune regulation [12,13], the function and biological signifi- cance of ISGylation remain elusive. However, recent evidences suggest an involvement of ISG15, in the form of conjugated protein modifiers, in regulation of IFN signaling and in antiviral immunity [9,14]. Both ubiquitin and ISG15 have been found to exist extracellularly in unconjugated forms. Extracellular ubiqui- tins are known to inhibit secretion of tumor necrosis factor PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e44884