As hypothesized, inhibiting SKI-1/S1P-mediated SREBP endoproteolytic cleavage events using Spn4A.RRLL(s) resulted in a dose-dependent decrease in HCV infection. We demonstrated that HCV core expression and HCV RNA levels are reduced in Spn4A.RRLL(s)-expressing cells following HCV infection, leading to a robust reduction in extracellular infectious HCV particle release. Western blotting also confirmed that HCV core protein post-translational processing was unaltered by serpin expression because the size of the protein was unaltered. Supplementing 1) The inactive SKI-1/S1P zymogen is biosynthesized in the ER and traffics to the Golgi apparatus following intramolecular autocatalytic maturation of the proenzyme [35, 46, 99, 100] . (2) During HCV infection, the SREBP pathway is activated by a variety of molecular mechanisms [19, [30] [31] [32] . (3) For SREBP to activate genes involved in lipid biosynthesis, its N-terminal domain must be released through sequential endoproteolytic cleavage first by SKI-1/S1P and then by S2P [37, 38] . (4) The released N-terminal domain translocates to the nucleus and activates various aspects of lipid metabolism [36] . (5) Activation of lipid biosynthesis increases LD formation where the HCV core protein localizes to orchestrate HCV assembly and subsequent secretion [8, 10, 16] . (6) Biosynthesis of LDLR, a proposed receptor for HCV entry, is also activated by SREBP signaling [69, 79, 101] . (7) Spn4A.RRLL(s) is a secretory pathwayexpressed serpin (Figure 1 ). (8) Spn4A.RRLL(s) interacts and forms a covalent complex with enzymatically active SKI-1/S1P molecules (Figure 2 and S3) in the Golgi apparatus preventing SKI-1/S1P-mediated endoproteolytic cleavage of SREBP protein ( Figure 3A ). (9) A small-molecule inhibitor PF-429242 also efficiently inhibits SKI-1/S1P endoproteolytic activity ( Figure 3A ). (10) SKI-1/S1P inhibition blocks expression of the putative HCV receptor, LDLR ( Figure 3B and S4) , and reduces HCV entry ( Figure 5) . (11) The expression of other SREBP-regulated genes, such as PCSK9 and SREBP-2, are also blocked ( Figure 3B ). (12) Downstream lipid synthesis is interrupted resulting in overall reduced intracellular cholesterol-ester and triglyceride abundance ( Figure 3C and 3D) . (12) This is then detected as a decrease in LD abundance (Figure 4) , which impedes assembly and secretion of infectious HCV particles. doi:10.1371/journal.ppat.1002468.g007