SUMMARY Human ISG15 is a ubiquitin-like protein (Ubl) that functions in innate immune responses, and it is remarkable for possessing three distinct biochemical activities. As a ubiquitin-like modifier it is conjugated to hundreds of cellular and viral proteins. The E1, E2, E3, and de-conjugating enzymes for ISG15 (Ube1L/Uba7, UbcH8/Ube2L6, Herc5, and Usp18, respectively), are, like ISG15, induced at the transcriptional level by Type I interferon (IFN-α/β) signaling. A second function of ISG15 is as an extracellular signaling protein. ISG15 is released into the extracellular space from many cell types and signals to Natural Killer and T cells to secrete IFN-γ, which plays a major role in the response to pathogen infections. The cell surface receptor for extracellular SG15 is LFA-1, an immune cell-specific integrin. A third function of human ISG15 is that it negatively regulates Type I IFN signaling, as revealed by the Type I interferonopathy that occurs in some ISG15-deficient patients; this function of human ISG15 is not shared with mouse ISG15. A challenge in the field is to understand how the activities of ISG15 are regulated and temporally controlled and which activities are most closely associated with responses to specific pathogens, including Mycobacterium tuberculosis and SARS-CoV-2. To further our understanding of ISG15 in innate immune responses to these and other pathogens, this proposal will focus on the basis of substrate and lysine selectivity of the Herc5/Herc6 family of ISG15 ligases, the ISG15-induced signaling pathway downstream of LFA-1 that leads to cytokine secretion, and the mechanism by which ISG15 released into the extracellular space.