It is now well established that hijacking of host-cell biosynthetic pathways by human enveloped viruses is a shared molecular event essential for the viral lifecycle [76] . The next frontier is identifying common, critical, host cell pathways that are hijacked by pathogenic human viruses, in order to develop broad-spectrum, host-directed antivirals with novel mechanisms of action. In this study, we hypothesized that targeting cellular enzymes acting as master regulators of lipid homeostasis could represent a powerful approach to developing a novel class of antiviral agents against infection associated with human enveloped viruses such as HCV, whose replication and pathogenesis depend on the interaction with lipid droplets (LDs) [8] . In the case of HCV, overstimulation of host lipid metabolism in the liver during viral infection promotes cholesterol intracellular storage in host LDs, a critical cellular event for the HCV lifecycle that leads to steatosis of the liver in HCV-infected patients [8, 18, 77] . One such master regulator of cholesterol metabolic pathways is the host proprotein convertase SKI-1/S1P [36, 78] . SKI-1/S1P plays a critical role in the proteolytic activation of SREBPs, which control expression of key enzymes of cholesterol and fatty-acid biosynthesis [41, 53] . Here, we report that strategic manipulation of cellular SKI-1/S1P activity levels by protein-based or small-molecule protease inhibitors provides a means of effectively microscope. (D) Huh-7.5.1 cells infected with Ad-Empty (control), Ad-Spn4A.RRLL(r), or Ad-Spn4A.RRLL(s) for 72 hours were harvested and subjected to SDS-PAGE and Western blot analysis. Mouse anti-ADRP antibody was used to detect protein expression levels in serpin-treated cells compared to control-treated cells. Relative protein expression was quantified by normalizing to b-tubulin expression. The inset shows a representative Western blot. (E) Huh-7.5.1 cells were treated with DMSO (control) or with 10 mM PF-429242 for 24 hours, the compound was removed, and the cell lysates were harvested after an additional 48 hours. Relative ADRP expression (normalized to b-tubulin) in inhibitor-treated cells compared to control cells was quantified by subjecting total cell lysates to Western blot analysis. Values are plotted relative to protein expression in control cells, which are set to 1. Results (mean 6 SEM) from 3 independent experiments are shown. *p,0.05. doi:10.1371/journal.ppat.1002468.g004 inhibiting HCV infection (JFH-1 strain) of Huh-7.5.1 cells in a dosedependent manner. Furthermore, we reveal the common molecular and cellular mechanisms of action of the SKI-1/S1P inhibitors and demonstrate that they act as negative modulators of cytoplasmic LD abundance (Figure 7) , an organelle central to HCV assembly [8] and liver steatosis.