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.

In vitro DHE and PI4P transport assays Previously described liposomal assays (Mesmin et al., 2013) were used to test the sterol and PI4P transfer activities of OSBP (schematically depicted in Figures 6A and 6B, respectively) . Briefly, sterol transfer is measured using the fluorescent cholesterol analog dehydroergosterol (DHE). DHE is transferred by OSBP from ER-like liposomes (ER like ) covered with VAP-A to Golgi-like liposomes (Golgil ike ) doped with dansyl-phosphatidylethanolamine (DNS-PE). Upon DHE transfer, there will be Förster resonance energy transfer (FRET) from DHE to DNS-PE due to the close proximity between the two fluorophores. In the PI4P-transfer assay, PI4P is transported by OSBP from Golgi-like liposomes to ER-like acceptor liposomes and detected using a sensor consisting of the FAPP1 PHdomain labeled with the fluorophore NBD ( NBD PH). When the sensor is bound to PI4P on the Golgi-like liposomes doped with rhodamine-PE (Rho-PE), NBD fluorescence is quenched by the rhodamine. Upon transfer of PI4P to the ER-like liposomes, the sensor moves from the Golgi-like to the ER-like liposomes and NBD-fluorescence is dequenched. Egg PC (L-α-phosphatidylcholine), liver PI (L-α-phosphatidylinositol), brain PS (L-αphosphatidylserine), brain PI4P (L-α-phosphatidylinositol-4-phosphate), Dansyl (DNS)-PE (1,2dioleoyl-sn-glycero-3-phosphoethanolamine-N-(5-dimethylamino-1-naphthalenesulfonyl)), Rhodamine (Rhod)-PE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)), DOGS-NTA-Ni 2+ (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]) were purchased from Avanti Polar Lipids. Cholesterol and dehydroergosterol (DHE) were from Sigma Aldrich. The concentration of DHE in stock solution in methanol was determined by UV-spectroscopy using an extinction coefficient of 13,000 M -1 .cm -1 . Full-length OSBP, VAP-A[8-212]His 6, NBD-PH FAPP1 and Arf1 were purified as described previously (Mesmin et al., 2013) . To prepare liposomes, lipids from chloroform solutions were mixed at the desired molar ratio, and the solvent was removed in a rotary evaporator. The lipid film was hydrated in 50 mM HEPES pH 7.2 and 120 mM potassium acetate (HK buffer) to give a suspension of large multilamellar liposomes. The suspension was then frozen and thawed five times and extruded through polycarbonate filters of 0.1 μm pore size using a mini-extruder (Avanti Polar Lipids). Unilamellar liposomes were stored in the dark and used within 2 days. For all transport experiment, ER-like liposomes contain: egg PC/brain PS/DOGS-NTA-Ni2 + (93/5/2 mol/mol) and Golgi-like liposomes contain: egg PC/liver PE/brain PS/liver PI/DNS-PE (63.5/19/5/10/2.5 mol/mol). Fluorescence experiments were performed in a Shimadzu RF-5301-PC spectrofluorimeter. The sample (volume 600 μl) was placed in a cylindrical quartz cell, continuously stirred with a small magnetic bar and equilibrated at 37°C. For DHE transfer assays, Golgi-like liposomes with 2.5 mol% DNS-PE (63.3 μM total lipids) were loaded with Arf1.GTP (0.3 μM) and incubated with 1 μM VapA[8-212]His 6 in HKM buffer (HK buffer supplemented with 1 mM MgCl 2 ) in the presence of 25-OH, ITZ or other azoles (different stock concentration in DMSO, DMSO/buffer final ratio v/v 1/100), prior to the addition of ER-like liposomes supplemented with 18 mol% DHE (63.3 μM total lipids) and of OSBP (100 nM final concentration). The sterol transport activity of OSBP was monitored by FRET be