ITRACONAZOLE INHIBITS ENTEROVIRUS REPLICATION BY TARGETING THE OXYSTEROL-BINDING PROTEIN

Strating, Jeroen R.P.M.; van der Linden, Lonneke; Albulescu, Lucian; Bigay, Joëlle; Arita, Minetaro; Delang, Leen; Leyssen, Pieter; van der Schaar, Hilde M.; Lanke, Kjerstin H.W.; Thibaut, Hendrik Jan; Ulferts, Rachel; Drin, Guillaume; Schlinck, Nina; Wubbolts, Richard W.; Sever, Navdar; Head, Sarah A.; Liu, Jun O.; Beachy, Philip A.; De Matteis, Maria A.; Shair, Matthew D.; Olkkonen, Vesa M.; Neyts, Johan; van Kuppeveld, Frank J.M.

radiol as in Figure 2 , and Renilla luciferase levels were measured after 6 hr. Scale bars correspond to 10 μm. Asterisks indicate statistical significance compared to the DMSO control. D, DMSO; Gu, Guanidine HCl (known replication inhibitor of CVB3); Posa, posaconazole; Keto, ketoconazole; Flu, fluconazole; Vori, voriconazole. (A) HeLa cells stably expressing the PI4P-sensor FAPP1-PH-GFP were treated with DMSO, 1 μM BF738735 (PI4KIIIβ inhibitor), or 10 μM ITZ for 2.5 hr, fixed and processed for microscopy. (B) HeLa R19 cells were treated as in (B), fixed and stained with an antibody against PI4P. (C) ITZ does not inhibit PI4P accumulation in infected cells. HeLa R19 cells were infected with CVB3 at MOI 10 and immediately after infection treated with DMSO or 10 μM ITZ, which does not fully inhibit replication. At 5 hr p.i., the cells were fixed and stained with antibodies against 3A and PI4P. Scale bars correspond to 10 μm. HeLa R19 cells were transfected with siRNA against OSBP or a scrambled siRNA as a negative control as in Figure 3D . After 48 hr, cells were fixed and immunostained for OSBP and GM130 as Golgi marker, and nuclei were counterstained with DAPI. Knockdown of OSBP at the protein level is evident by a weaker OSBP staining both on Golgi structures and throughout the cytoplasm. Of note, OSBP knockdown caused the Golgi to become less compact, which at the same time demonstrates that knockdown was efficient enough to induce a physiological effect. The scale bar corresponds to 10 μm. Huh 9-13 cells carrying a HCV genotype 1b subgenomic replicon were treated with ITZ or other antifungal azoles and replication was determined after 3 days by Q-PCR. Values are plotted as % of untreated control (UTC), experiments were performed in triplicates and mean ± SEM are plotted. EC50 values are means ± SD from an experiment performed in triplicate. For all compounds, 50% cytotoxic concentrations (CC50) were >30 μM (data not shown).