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.

Target identification by siRNA sensitization (TISS) assay TISS assay was performed as previously described (Arita et al., 2011) . In short, HEK293 cells in 96well plates were transfected with siRNA pools by using Lipofectamine RNAiMAX transfection reagent (Invitrogen). At 72 hr post transfection, these cells were infected with 800 infectious units PV pseudovirus and treated with DMSO or 1.25 μM ITZ. Firefly luciferase activity was measured at 7 hr p.i. using Steady-Glo Luciferase Assay System (Promega). The effect of siRNA treatment on the sensitivity to each compound was determined by calculating the normalized PV pseudovirus infection. This number represents the level of firefly luciferase activity for compound-treated and siRNAtransfected cells divided by the firefly luciferase activity measured in siRNA-transfected cells in the absence of compounds.