We selected a Drosophila serpin, Spn4A, as a prototype macromolecular inhibitor scaffold. The identification of Spn4A by our group as the most potent natural serpin inhibitor of the human PC furin (K i ,13 pM [58] ) provides us with a novel and unique molecular tool for dissecting the contribution of SKI-1/ S1P-dependent proteolytic activity in the secretory pathway to viral infection of eukaryotic cells. We report that engineering of the RCL of Spn4A to mimic the consensus sequence Arg/Lys P4 -Xaa 3 -Xaa 2 -Leu/Ser/Thr P1Q for cleavage by SKI-1/S1P (Spn4A RCL: Arg P4 -Arg-Lys-Arg P1Q -. Arg P4 -Arg-Leu-Leu P1Q ) resulted in the development of Spn4A-RRLL, a novel, selective, and effective serpin-based inhibitor of SKI-1/S1P. We demonstrated the antiproteolytic and anti-HCV activities of our new recombinant adenovirus-expressing Spn4A-RRLL ''secreted'' (s) variant directed at the secretory pathway SKI-1/S1P. Expression of Spn4A.RRLL(s) in Huh-7.5.1 cells results in a strong inhibition of the SKI-1/S1P-mediated activation of SREBP-1 and downregulation of SREBP target gene products. As hypothesized, inhibiting SKI-1/S1P activity robustly blocked HCV infection of Huh-7.5.1 cells in a dose-dependent manner. We found that specific inhibition of SKI-1/S1P activity by Spn4A.RRLL(s) dramatically reduced the abundance of LDs in hepatoma cells. Use of the specific active site-directed small-molecule inhibitor of SKI-1/S1P, PF-429242, confirmed the results of our studies with the protein-based therapeutic Spn4A.RRLL(s), with a robust inhibition of HCV infection.