Atlantic salmon ISG15: Expression and conjugation to cellular proteins in response to interferon, double-stranded RNA and virus infections

ISG 15 is one of the earliest and most predominant proteins to be induced in mammals following IFN-alpha/beta stimulation, which suggests that it has an important function in the interferon system. Similar to ubiquitin, ISG15 forms covalent conjugates with its target proteins, but free ISG15 is released from human lymphocytes and monocytes during IFN-alpha/beta stimulation. In this work we describe a 17.3 kDa ISG15 orthologue in Atlantic salmon (AsISG15) with characteristic features of ISG15 proteins including tandem ubiquitin-homology domains and a conserved carboxy-terminal conjugating motif (LRLRGG). Furthermore, Northern blot analysis revealed strong induction by polyinosinic polycytidylic acid (poly I:C) and by viral infections, while Western blot analysis using a specific antibody generated against AsISG15 confirmed induction mediated by recombinant Atlantic salmon IFN-alpha/beta and demonstrated conjugation of AsISG15 to cellular proteins. Interestingly, the pattern of AsISG15 modified target proteins differed during ISAV infection compared to direct IFN-alpha/beta stimulation. Immunoprecipitation experiments demonstrated extracellular, free AsISG15 in supernatants of leucocytes stimulated with poly I:C. Moreover, immunoprecipitation of an about 65 kDa ISAV protein from infected TO cells using anti-AsISG15 antiserum suggests that binding between the AsISG15 and the ISAV protein occurred. Taken together, the results suggest that AsISG15 has a role in the antiviral interferon response of Atlantic salmon. (c) 2006 Elsevier Ltd. All rights reserved.