Research Article: Cynoglossus semilaevis ISG15: A Secreted Cytokine-Like Protein That Stimulates Antiviral Immune Response in a LRGG Motif-Dependent Manner

Date Published: September 18, 2012

Publisher: Public Library of Science

Author(s): Wei Wang, Min Zhang, Zhi-zhong Xiao, Li Sun, Hidde L. Ploegh.


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.

Partial Text

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].

In this study, we identified CsISG15, an ISG15 homologue from tongue sole, and analyzed its expression and function. Like all known ISG15, CsISG15 possesses two tandem UBL domains and the highly conserved LRGG motif, which categorize CsISG15 as a member of the ISG15 family. In fish, reports have shown that expression of most of the identified ISG15 is enhanced by microbial challenge. For example, viral infection and poly(I:C) treatment activate the expression of the ISG15 of Atlantic cod [28], [29], [31], Atlantic salmon [27], [32], black rockfish [21], channel catfish [23], crucian carp [26], and Japanese flounder [25], while bacterial challenge activates ISG15 expression in Atlantic cod [25], goldfish [22], and red drum [30]. In this study, we observed constitutive CsISG15 expression in a wide range of tissues and drastically enhanced expression of CsISG15 in kidney and spleen following experimental infection with megalocytivirus and V. anguillarum. In both kidney and spleen, the induction patterns caused by viral and bacterial challenges differed, probably due to different activation mechanisms effected by these two pathogens. Consistent with these observations in a live fish model, cellular study showed that megalocytivirus infection induced strong induction of CsISG15 expression in HK lymphocytes in a time-dependent manner. Like human ISG15, which is known to be secreted into the extracellular milieu, CsISG15 protein was detected in the culture medium of HK lymphocytes following viral challenge. These results indicate that viral challenge stimulates CsISG15 expression at both transcription and translation levels. Since, like all ISG15, CsISG15 lacks an apparent signal sequence, extracellular exportation of CsISG15 is probably accomplished through a non-classical secretion pathway.