If elimination of UBP43 is shown to reduce viral replication or magnify the interferon response, we hypothesize that inhibition of UBP43 will represent a novel means of treatment of HCV. UBP43 would be of particular interest because it has protease activity: proteases are known and validated targets for small molecule therapies. Thus, it would be critical to establish whether the protease activity of UBP43 is important for its effect on HCV replication, and not, for instance, its interaction with another cellular protein. To address this question we will over-express UBP43 protease inactive constructs. An inactive form of UBP43 will be created by mutation of a critical cysteine residue (Cys61) into serine using site-directed mutagenesis of pBK/CMV-UBP43 plasmid as described previously. In order to characterize the protease active and inactive forms of UBP43, wild type and mutant UBP43 will be expressed as GST fusion proteins in the expression vector pGEX-4T-3 (pGEX-4T-3-UBP43), and transformed into E.Coli BL21 (DE3) with a plasmid, pET-ISG15-UBP43-H, that expresses ISG15-UBP43 fusion protein.35 ISG15 cleavage from the fusion protein will be shown by Western blot. Once we have confirmed that the protease function is eliminated by site-directed mutagenesis, wild-type and mutated UBP43 will be overexpressed in Huh7.5 cells with the HCV replicon and exposed to increasing doses of IFNa. If the protease function is critical overexpression of the inactive (in a dominant negative fashion) will increase responsiveness to IFN.