Determinants of ISG15 secretion and extracellular signaling

ISG15 is a ubiquitin like protein that also functions as an extracellular cytokine. In response to type I interferon, ISG15 is expressed and conjugated to newly synthesized proteins in a three-enzyme trans-thiolation cascade (ISGylation). The E1, UBE1L, activates ISG15, transfers it to the E2, UBCH8, which then transfers it to the E3 HERC5 (HERC6 in mice). HERC5 associates with ribosomes and ISGylates primary amines cotranslationally. This is thought to be antiviral by inhibiting oligomerization of viral protein complexes. Interestingly, ISG15 has a second function as an extracellular cytokine. In response to bacterial and viral Pathogen Associated Molecular Patterns (PAMPS) and mycobacterial infections, ISG15 induces IFN-γ secretion. I showed that specific residues of ISG15 are required for secretion of ISG15 from cells (L72, S83, and L85), and that residues Y96, R99, T101, Q102, and T101 are required for extracellular signaling. I identified the LFA-1 integrin as the ISG15 receptor, and showed that ISG15 stimulates NK and T cells to promote the secretion of IFN-γ, IL10 and IL6. Viral pathogens have evolved mechanisms to modulate ISG15 secretion. Influenza B NS1 binds ISG15 and I showed that this interaction prevents ISG15 secretion. Other viruses like SARS-CoV-2, ERVE and FMDV express enzymes that reverse ISG15 conjugation creating free ISG15 and I showed that de-ISGylation enhances ISG15 secretion. These viral de-ISGylates are good targets for anti-viral drug development. I showed that the FDA approved drug, 6-thioguanine, is a small molecule inhibitor of the SARS-CoV-2 de-ISGylase (PLpro) that blocks both viral polyprotein cleavage and de-ISGylation and inhibits viral replication as sub-micromolar concentrations. It is becoming clear that the seemingly disparate functions of ISGylation and ISG15 secretion are related and interact to promote a robust immune response.