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.

Subgenomic replicon assays for CVB3 and PV were performed as described previously using the replicons pRib-LUC-CB3/T7 and pPV-Fluc mc (wt and 3A[A70T]) respectively (Aminev et al., 2003; Arita et al., 2009; Van Ooij et al., 2006) . In these subgenomic replicons the P1 area was partially (EMCV) or completely (CVB3 and PV) replaced by the sequence encoding the firefly luciferase. Linearized plasmids of the replicon constructs or of pRLuc-QG-M16.1 (EMCV infectious clone) were in vitro transcribed using T7 RNA polymerase. RNA was transfected into cells using either DEAE-dextran or Lipofectamine RNAiMAX and firefly (CVB3, PV) or Renilla (EMCV) luciferase activity was analyzed using the Luciferase Assay System (CVB3, PV) or the Renilla Luciferase Assay System (EMCV) (Promega) at the indicated time points. Hepatitis C virus antiviral assays were performed using previously described Huh-7 cells containing subgenomic HCV replicon I 377 /NS3-3'/wt (Huh 9-13) (Lohmann et al., 1999) . Cells were cultured in DMEM (Gibco) supplemented with 10% heat-inactivated FCS (Integro, The Netherlands), 1x nonessential amino acids, 100 IU/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco) and 1 mg/ml Geneticin ® (G418; Gibco). Antiviral assays were performed as described before (Delang et al., 2012) .