Previous work using thymic tissue has identified several targets for ISGylation by ISG15, including phospholipase Cg1, Jak1, and ERK1. Whether these are targets in Huh7.5 cells is not known, though ERK and JAK have been previously linked to antiviral effects in the HCV replicon model. If we find that ISG15 is implicated in HCV replication, we would seek to identify substrates for ISG15 modification. We will first perform a series of immunoprecipitation studies directed specifically at the proteins previously shown found to be modified by ISG15, and explore the differences between infected and uninfected cells. In brief, following incubation of Huh7.5 cells +/- replicon with IFNa for 12 hours, cells will be lysed in RIPA buffer and immunoprecipitation of ISG15 conjugates performed with the anti-ISG15 mAb used above. After resolution on a reducing gel, western blots will be performed to determine if the three targets above are conjugated by ISG15, using commercially-available mAbs. If any or all of the targets are conjugated, further studies will be performed to elucidate the consequences of ISGylation.