As described earlier, cells expressing the HCV polyprotein contained significantly elevated amounts of intracellular PI4P, which were reduced dose dependently AG-014699 mw by BMS-553. This reduction was not observed with the Y93H resistance mutant ( Figure 3D), pointing to specific inhibition of NS5A-dependent activation of PI4KIIIα by BMS-553. Therefore, potent NS5A inhibitors reduce NS5A-mediated intracellular accumulation of PI4P, which might be due in part to impaired NS5A-PI4KIIIα interaction. 31 Given the important role of NS5A and PI4KIIIα for MW formation and morphology,6, 7 and 31 we examined HCV-induced membrane

alterations in cells expressing either wt

or Y93H-containing NS3-5B polyprotein after BMS-553 treatment. Of note, this expression system induces membrane rearrangements well, comparable with those detected in cells containing a functional HCV genome (Supplementary Figure 10).6 Treatment of polyprotein-expressing cells from 6 hours post transfection, referred to as “posttreatment,” affected neither NS5A expression (Figure 1D, right panel) nor the number of NS5A-expressing cells ( Supplementary Figure 11A). Electron microscopy analysis of mock-treated INCB024360 cells revealed regular MW structures, most notably double membrane vesicles (DMVs), the major MW constituents and possible sites of HCV RNA replication 6 ( Figure 4A, top left

image). Upon treatment with BMS-553, the MW collapsed concentration dependently and, after high-dose treatment, only web remnants were detectable in few cells ( Figure 4A). Smoothened In contrast, web morphology was unaffected in BMS-553–treated cells expressing the Y93H-containing polyprotein ( Figure 4A, middle column), thus excluding pleiotropic or cytopathic effects as a reason for web inhibition in wt polyprotein-expressing cells. In case of the wt polyprotein DMV diameter was reduced dose dependently ( Figure 4B), resembling the phenotype we had observed earlier upon PI4KIIIα knock-down 7 or upon treatment with the PI4KIIIα inhibitor AL-9 32 ( Figure 4A and B). Importantly, DMV number was also massively reduced, both by BMS-553 and AL-9. In contrast, in cells expressing the Y93H mutant, DMV diameter was slightly increased and DMV number was not affected. An even more striking effect was found with BMS-553 treatment, starting at the time point of transfection (referred to as co-treatment). Again, NS5A expression per se, as well as the number of NS5A-expressing cells, was not affected (Figure 1D and Supplementary Figure 11B).