Structural aspects of the ISGylation process: Impact on the innate immune response and ribosome function

The innate immune response is the first line of defense against viral pathogens. The response is non-specific and incorporates the secretion of cytokines like IFN-I. One of the primary IFN induced genes (ISGs) is ISG15, a small IFN-I-induced ubiquitin-like protein, which rapidly modifies a vast array of cellular and viral targets via a mechanism known as ISGylation. ISGylation is mediated via an E1-E2-E3 enzymatic cascade, like ubiquitination, but to date, there are only three known ISG15-E3 ligases TRIM25, HHARI, and HERC5 that catalyze the final transfer to target proteins, resulting in substrate modification. The ISGylation machinery, and HERC5 specifically, have been linked to ribosomes, suggesting ISG15 preferably modifies newly synthesized proteins. However key questions remain unanswered: Do all ISG15-E3 ligases act in a co-translational manner? What is the co-translational substrate pool? What is ISGylation`s impact on the fate of the nascent proteome of the host? What domains confer ribosome binding? To answer these questions, we mechanistically analyze ISGylation at the ribosome, using polysome and ribosome profiling approaches to elucidate the function of the ISG15-E3 ligases in the innate immune response. In addition, structural and functional analyses of the ISG15-E3 ligases were done using AlphaFold2 generated models to predict the E3s-ISG15 complexes and RNA binding sites, elucidating the interplay between ISG15, nascent chains, and ribosomes. Model-based predictions show that TRIM25 and HHARI both have possible rRNA binding sites, indicating that ISGylation may be a co-translational modification. Current ribosome profiling analysis will provide crucial evidence for these predictions.