3 μM for BIT225, NN-DNJ and Rimantadine, respectively, compared to IC90 values of 30, 30 and 1 μM for SA13/JFH (data not shown). The higher IC90 values reported here compared to previous studies most likely

reflect differences in the duration of treatment, with earlier studies treating infected cells for up to 72 h before measuring extracellular virus infectivity. Since Volasertib datasheet NN-DNJ can affect glycosylation of viral proteins we limited the duration of treatment to minimise such off-target effects. The efficacy of the inhibitors to limit HCV cell-to-cell transmission was tested using a recently developed single-cycle co-culture assay (Meredith et al., 2013). Since p7 has been reported to play a role in viral internalisation (Griffin et al., 2008) it is important to discriminate the effect of p7 inhibitors on virus assembly and entry. This assay allows one to assess the effect of p7 inhibitor treatment on infected ‘producer’ cells and enables the quantification of new infection events within 2 h of culturing infected and naïve hepatoma cells, which is essential given the reversible nature of p7 targeted compounds NLG919 molecular weight (Pavlovic et al., 2005 and Pavlovic et al., 2003). HCV J6/JFH or SA13/JFH infected Huh-7.5 cells were treated with 30 μM of either BIT225 or NN-DNJ and 3 μM Rimantadine for 24 h, concentrations previously shown to inhibit the level of infectious extracellular virus by 80–90%. The cells were washed to remove the compounds, labelled

with 5-Chloromethylfluorescein diacetate (CMFDA Cell Tracker Green, Invitrogen), and cultured with naïve Huh-7.5 targets at a 1:1 ratio as detailed in Fig. 1A. We confirmed that all compounds reduced the level of extracellular infectious virus in the co-culture ( Fig. 1B and C), consistent

with a reduction in J6/JFH and SA13/JFH cell-free transmission events. Although all three compounds inhibited 50–70% of J6/JFH cell-to-cell transmission, they had no detectable effect on SA13/JFH cell-to-cell transmission ( Fig. 1C). To determine how wide ranging this effect was, we screened a panel of diverse chimeric viruses expressing the structural proteins from genotype 1–7 for their sensitivity to all currently available p7 inhibitors, including NN-DGJ that does not affect host cell glycosylation pathways ( Chapel et al., 2006a, Chapel et al., 2006b and Chapel et al., 2006c). 4-Aminobutyrate aminotransferase Three viruses (JFH-1 – GT2; ED43/JFH – GT3 and QC69/JFH – GT7) showed limited transmission and were excluded from the analysis. The results show that all of the p7 inhibitors were significantly more effective at inhibiting cell-free infection than cell-to-cell transmission when all genotypes are considered ( Fig. 1D). The recent study by OuYang et al., suggest that amantadine binds the p7 ion-channel and locks it into a closed position (OuYang et al., 2013), preventing the de-acidification of the intravesicular compartments and leading to the secretion of non-infectious virus.