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.

PH-FFAT (OSBP fragment 76-408) was purified as described (Mesmin et al., 2013) , and the flotation experiments were done as previously detailed (Bigay et al., 2005) . Briefly, PH-FFAT (0.75 μM) was incubated 5 min at room temperature with 0.75 mM liposomes (ER-or Golgi-like as prepared for the PI4P transfer assay), with or without VAP-A (2 μM) and/or ITZ (1 or 10 μM) as indicated. The suspension (150 μl) was mixed with 100 μl high sucrose solution (30% w/v final sucrose concentration), and overlaid by 200 μl 25% w/v sucrose solution, then by 50 μl buffer without sucrose. The sample was centrifuged at 55,000 rpm in a TLS-55 rotor (Beckman) for 1 hr at 20°C. The bottom (250 μl), middle (150 μl) and top (100 μl) fractions were manually collected from the bottom with a Hamilton syringe. Proteins bound to floating liposomes present in top fractions were analyzed by SDS-PAGE.