Interferon-stimulated gene 15 (ISG15) is a 17 kDA secreted protein that in humans is encoded by the ISG15 gene. ISG15 is induced by type I interferon (IFN) and serves many functions, acting both as an extracellular cytokine and an intracellular protein modifier. The precise functions are diverse and vary among species but include potentiation of Interferon gamma (IFN-II) production in lymphocytes, ubiquitin-like conjugation to newly-synthesized proteins and negative regulation of the IFN-I response. Structure The ISG15 gene consists of two exons and encodes for a 17 kDa polypeptide. The immature polypeptide is cleaved at its carboxy terminus, generating a mature 15 kDa product that terminates with a LRLRGG motif, as found in ubiquitin. The tertiary structure of ISG15 also resembles ubiquitin, despite only ~30% sequence homology. Specifically, this structure consists of two ubiquitin-like domains connected by a polypeptide ‘hinge.’ Of note, ISG15 shows substantial sequence variation among species, with homology as low as 30% between orthologs. Function After induction by type I interferon, ISG15 can be found in three forms, each with unique functions: Extracellular cytokine ISG15 is secreted from the cell and can be detected in supernatant or blood plasma. ISG15 binds the LFA-1 integrin receptor on NK- and T-cells to potentiate their production of IFN-II, which is essential for mycobacterial immunity. Intracellular conjugate: ISGylation In a ubiquitin-like fashion, ISG15 is covalently linked by its C-terminal LRLRGG motif to lysine residues on newly synthesized proteins. This process, termed ISGylation, is catalyzed by a series of conjugating enzymes. The activating E1 enzyme (UBE1L) charges ISG15 by forming a high-energy thiolester intermediate and transfers it to the UbcH8 E2 enzyme. UbcH8 has been identified as the major E2 for ISGylation, although it also functions in ubiquitination. The E2 protein subsequently transfers the ISG15 to specific E3 ligases (Herc5) and relevant intracellular substrates. Only one deconjugating protease with specificity to ISG15 has been identified to date: USP18 (a member of the USP family) cleaves ISG15-peptide fusions and also removes ISG15 (deISGylation) from native conjugates. The effects of ISGylation are incompletely understood and involve both activation and inhibition of antiviral immunity. Free intracellular molecule Unconjugated ISG15 negatively regulates IFN-I signaling by preventing the SKP2-mediated proteasomal degradation of USP18, a direct inhibitor of the IFN-I receptor. Absence of ISG15 leads to persistent IFN-I signaling in human, but not mouse, systems. Clinical significance ISG15-deficiency is a very rare genetic disorder caused by mutations of the