Although ISG15 is the first UBL known to exist, its biological role and mechanism of action are less well understood than most of the other UBLs, such as SUMO or NEDD8. This is in part due to the absence of homologues of ISG15 and its conjugation machinery (e.g, UBE1L) in experimental organisms such as yeast, Drosophila or C. elegans. Nevertheless, significant progress has been made in the past few years in the identification of the enzymatic machinery that carries out ISGylation and in the elucidation of the role of ISGylation in antiviral defense. The recent findings of the direct antiviral activity of ISG15 through both specific and broad modification of viral proteins represent a major advance in understanding the antiviral mechanisms of ISGylation. Some ISGylated host proteins also appear to mediate its antiviral effects (for example ISGylated 4EHP and IRF3 as mentioned above).
Although upregulating the expression of ISGylation machinery is a primary means of regulating ISGylation, additional regulatory mechanisms clearly exist, for example, NS1B’s binding to ISG15 and HERC5’s association with ribosomes and specific substrates like NS1A. Biochemical reconstitution of the ISGylation process would potentially facilitate the identification of additional factors that regulate ISGylation.
An emerging theme from the recent mechanistic studies is that ISGylation alters a protein’s ability to engage in its typical interactions (such as with other proteins or RNA). The basis for this alteration is as yet unclear. It is likely that the presence of ISG15 could directly interfere with the normal protein-protein or protein-RNA interface. It is also feasible that ISGylation could induce allosteric changes in protein structure, or that ISG15-binding protein(s) may be present in cells and could modulate interactions between ISGylated proteins and their typical partners.
It is noteworthy that mice lacking ISG15 are not as susceptible to viral infection as IFN receptor knockout mice, indicating that ISGylation contributes to, but is not solely responsible for, the antiviral effects of IFN in mice (Lenschow et al., 2007
). Recent work demonstrating marked differences in the interaction between influenza B virus and the ISGylation machinery of mice and humans suggests that ISG15 might play a more prominent antiviral role in human. Indeed, blocking ISGylation in human cells severely impairs IFN-induced antiviral activity against Influenza A virus (Hsiang et al., 2009
). Future research could also reveal other functions of ISGylation unrelated to its antiviral effect. Indeed, the levels of ISG15 and its conjugation to cellular proteins are elevated in several tumors and tumor-derived cell lines (Desai et al., 2006
Understanding the roles and mechanism of action of ISGs, such as ISG15, in antiviral defense may pave the way to more effective antiviral therapies. For example, viral proteins that counter the IFN response by antagonizing ISGylation might make appealing therapeutic targets.