Factors that balance free and conjugated ISG15 during the immune response. 1. Binding of interferon (IFN) to the interferon receptor (IFNR) results in a conformational change that enables the transphosphorylation of associated Janus Kinase (JAK). The activated JAK then phosphorylates adjacent signal transducer and activator of transcription (STAT) proteins. 2. Phosphorylated STAT dimers migrate to the nucleus where they bind to interferon-sensitive response elements (ISRE) inducing hundreds of interferon-stimulated genes (ISGs), among them are ISG15, ISG15 conjugating enzymes (e.g. UBE1L, UBE2L6, HERC5), and its specific deISGylase (DIG), USP18). 3. HERC5, the major ISG15 E3 ligase may interact with the ribosome where it massively labels synthesized proteins (from both host or viral transcripts) as they emerge from the ribosome (3a). An as-yet-unidentified E3 may interact with a ubiquitous protein signal such as ubiquitin to generate mixed Ub-ISG15 modifications on target proteins (3b). 4. ISGylation contributes to innate immune response and inhibits viral replication, though the precise mechanism remains elusive. 5. ISGylation is overturned by USP18, a DIG that can remove ISG15 from a substrate releasing free ISG15. 6. The free form of ISG15 can be conjugated to new targets (by HERC5 and other E3 ligases), or stabilize USP18, a known inhibitor of the STAT-JAK pathway (unrelated to its catalytic DIG activity) thereby inhibiting the innate immune response. Free ISG15 can also be secreted from the cell and act as a cytokine-like protein thereby enhancing the host immune response. 7. Some viruses evolved the ability to bypass the ISG15 pathway. Coronaviruses encode a PLpro enzyme that can cleave Ub or ISG15 from conjugates (in addition to cutting the viral polypeptide precursors, PP1a, PP1ab, into an array of functional proteins (NSPs) initiating the replication of the virus). 8. By counteracting the conjugating pathway, the virus generates free ISG15. |Deconjugating ISG15 may benefit the virus to bypass the host's innate immune response, but the formation of free ISG15 could also alert neighboring cells (6).