Next, we wanted to test whether extracellularly applied PF-429242 would effectively inhibit the endoproteolytic activity of secretory pathway SKI-1/S1P and reduce HCV infection in Huh-7.5.1 cells. First, using an MTS-based cell viability assay, we confirmed that no major cytotoxic effects occur in Huh-7.5.1 cells treated with up to 50 mM of PF-429242 ( Figure S6 ). Next, cells were treated with increasing concentrations (0.05 mM to 50 mM) of PF-429242 for 24 hours before the cell media was replaced and cells were infected for 48 hours with HCV. The number of HCVinfected cells, indicated by positive core protein expression, was measured using Cellomics HCS ( Figure 6A Because PF-429242 and Spn4A.RRLL(s) decrease the abundance of LD components, we hypothesized that HCV assembly, rather than HCV replication, can also blocked by SKI-1/S1P inhibition. To support this hypothesis, Huh.8 and Huh.2 cells were treated with 10 mM PF-429242 for 72 hours. No significant changes in HCV subgenomic RNA levels in either cell line ( Figure 6C ) treated with PF-429242 were observed (compared to DMSO-treated control). In summary, with the lack of effect of PF-429242 on HCV replicon levels and comparing the two sets of data presented in Figure 6B , which demonstrate a strong antiviral effect of PF-429242 when added either pre-or post-HCV inoculation, we can propose that pharmacological inhibition of SKI-1/S1P endoproteolytic activity by PF-429242 impacts late assembly stages of the HCV lifecycle.