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.

Microscopic analyses were performed using HeLa R19, BGM or RD cells grown to subconfluency on coverslips in 24-well plates. Where indicated, cells were transfected with plasmids FAPP1-PH-GFP, pEGFP-OSBP, or pEGFP-Golgi (encoding GFP-fused GalT aa1-81; Clontech) using Fugene (Roche) according to the manufacturer's instructions, and after overnight expression the cells were treated with compounds as indicated. In other experiments, cells were infected with CVB3 and in some cases treated with compounds as indicated. Cells were fixed at the indicated time points after addition of the drugs or after infection with 4% paraformaldehyde, permeabilized with PBS/0.1% Triton X-100 (for PI4KIIIβ stainings) or PBS/0.2% saponin/5% BSA (all other experiments), immunostained with antibodies, in some cases DNA was counterstained with Hoechst-33258 or DAPI, and embedded in Mowiol (PolySciences) or FluorSave (Merck). For Filipin staining, cells were fixed and permeabilized with saponin as above, then stained with 25 μg/ml Filipin III (freshly diluted from a 25 mg/ml stock in DMSO) (Sigma) in PBS and embedded in FluorSave. Cells were imaged using standard Leica DMR or Olympus BX60 microscopes, a Leica SPE-II DMI-4000 confocal laser scanning microscope, or a Nikon Ti Eclipse microscope equipped with an Endor DU/897 EMCCD-camera. To analyze co-localization of OSBP with 3A, images were processed using ImageJ as follows. The a background signal derived from an area without cells was subtracted from the image, single cells were outlined and a mask was created, and all signal outside the mask was cropped to exclude it from the calculations. Manders' co-localization coefficient was calculated using the JACoP plugin (Bolte and Cordelieres, 2006) with a manually set threshold.