Itraconazole inhibits enterovirus replication by targeting the oxysterol-binding protein

Abstract

Itraconazole (ITZ) is a well-known antifungal agent that also has anticancer activity. In this study, we identify ITZ as a broad-spectrum inhibitor of enteroviruses (e.g., poliovirus, coxsackievirus, enterovirus-71, rhinovirus). We demonstrate that ITZ inhibits viral RNA replication by targeting oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4). Consistently, OSW-1, a specific OSBP/ORP4 antagonist, also inhibits enterovirus replication. Knockdown of OSBP inhibits virus replication, whereas overexpression of OSBP or ORP4 counteracts the antiviral effects of ITZ and OSW-1. ITZ binds OSBP and inhibits its function, i.e., shuttling of cholesterol and phosphatidylinositol-4-phosphate between membranes, thereby likely perturbing the virus-induced membrane alterations essential for viral replication organelle formation. ITZ also inhibits hepatitis C virus replication, which also relies on OSBP. Together, these data implicate OSBP/ORP4 as molecular targets of ITZ and point to an essential role of OSBP/ORP4-mediated lipid exchange in virus replication that can be targeted by antiviral drugs.

Graphical abstract

Figure 1

ITZ Inhibits Viruses at the Genome Replication Stage (A) BGM (CVB3, EV71, EMCV, ERAV) or HeLa R19 cells (HRV14, SAFV) were infected with virus at multiplicity of infection (MOI) 1 and treated with ITZ. Virus titers at 8 hr postinfection (p.i.) (10 hr for SAFV) were determined by endpoint dilution. (B) Cell viability with MTS assay after 8 hr incubation with ITZ. (C) BGM cells were transfected with RNA of subgenomic replicons pRib-LUC-CB3/T7 or pRLuc-M16.1 (EMCV) and treated with DMSO, 25 μM ITZ, or as positive controls 2 mM GuHCl or 80 μM dipyridamole, and luciferase levels were determined at the indicated time points. Experiments were performed in triplicate and mean values ± SEM are shown; asterisks indicate statistical significance compared to mock treated controls. See also Figures S1 and S2.

Figure 2

ITZ Does Not Inhibit Virus Replication through Known Targets or PI4KIIIβ, although CVB3 with Mutations in the Nonstructural Viral Protein 3A Are Cross-Resistant to ITZ and PI4KIIIβ Inhibitors (A, B, and D) HeLa R19 (A) or BGM (B and D) cells were infected with RLuc-CVB3 at MOI 0.1 and treated with 10 μM ITZ, DMSO, or 10 μM antifungal azoles (A), Hedgehog pathway antagonists (100 nM Sant-1, Sant-2, or cyclopamine-KAAD) (B), or ERα (β-estradiol) (D), and Renilla luciferase levels were measured after 6 hr. (C) HAP1 cells were treated with 10 μM antifungal azoles for 6 hr and fixed, and cholesterol was stained with filipin. (E) BGM cells were infected, treated, and analyzed as in (A) with RLuc-CVB3 WT or the 3A[H57Y] mutant. (F) BGM cells were infected with CVB3 WT or CVB3 3A[H57Y] at low MOI in the presence of ITZ, and cell viability was measured after 3 days. (G and H) In vitro-transcribed RNA of subgenomic replicons pRib-LUC-CB3/T7 (WT and indicated 3A mutants) (G) or pPV-FLuc (WT and 3A[A70T]) (H) was transfected into RD cells. The cells were treated with DMSO, 25 μM ITZ, or 1.5 μM T-00127-HEV1 (PI4KIIIβ inhibitor), and firefly luciferase levels at 7 hr p.i. were determined. (I) HeLa R19 cells were transfected with FAPP1-PH-GFP treated with DMSO, 25 μM ITZ, or 1 μM PIK93 for 1 hr and stained with an antibody against PI4KIIIβ and Hoechst. Experiments were performed in triplicate and shown are mean values ± SEM; asterisks indicate statistical significance compared to mock-treated controls (A and B) or of mutant virus compared to WT. Scale bars correspond to 10 μm. See also Figures S3 and S4.

Figure 3

ITZ Inhibits Virus Replication by Targeting OSBP and ORP4 (A) HEK293 cells were transfected with siRNAs targeting PI4P-binding proteins, infected with PV, and incubated in the presence of 1.25 μM ITZ. Normalized PV infection represents the level of firefly luciferase activity at 7 hr p.i. for siRNA-transfected and compound-treated cells divided by the firefly luciferase activity measured in siRNA-transfected and untreated cells. (B) HeLa R19 cells were infected with RLuc-CVB3 WT or the 3A[H57Y] mutant at MOI 0.1 and treated with OSW-1, and Renilla luciferase levels were determined after 7 hr. Cell viability was determined in parallel. (C) HAP1 cells were treated for 6 hr with 10 nM OSW-1 or 10 μM ITZ, fixed, and stained with filipin. (D) HeLa R19 cells were transfected with constructs encoding OSBP or EGFP (negative control) for 24 hr, infected with RLuc-CVB3 at MOI 0.25 or EV71 at MOI 1, and treated with 10 μM (CVB3) or 3 μM (EV71) ITZ, 3 nM OSW-1, or DMSO. Renilla luciferase levels were determined at 7 hr p.i. (CVB3) or virus titers at 10 hr p.i. were determined by endpoint titration (EV71). (E) HeLa R19 cells were transfected with siRNAs against OSBP, PI4KIIIβ (positive control), or a scrambled siRNA for 2 days and infected with CVB3, EV71, or HRV2 at MOI 1. Virus titers at 10 hr p.i. were determined by endpoint titration. Knockdown efficiency was determined by quantitative PCR and immunofluorescence (Figure S5), and an MTS assay was used to test for effects on cell viability. (F) HEK293 cells were transfected with siRNAs targeting ORP family members (roman numbering indicates ORP subfamilies), infected, treated with ITZ, and analyzed as in (A). (G) HeLa R19 cells were transfected with constructs encoding OSBP, ORP4, or enhanced GFP, infected and treated with 3 nM OSW-1, and data were analyzed as in (C). All figures are representative examples of experiments that were performed in triplicate. Shown are mean values ± SEM. Scale bars correspond to 10 μm. See also Figures S5 and S6.

Figure 4

Azoles that Inhibit Virus Replication Rapidly Accumulate OSBP at the Golgi (A) HeLa R19 cells were transfected with OSBP-GFP; treated with DMSO, 10 μM of ITZ or antifungal azoles, or 10 nM OSW-1 for 1 hr; and fixed and counterstained with an antibody against PI4KIIIβ and DAPI. (B) HeLa R19 cells were treated as in (A), fixed and immunostained for endogenous OSBP. (C) HeLa R19 cells were transfected with GFP-OSBP and treated with DMSO, 10 μM of ITZ, or 10 nM OSW-1, and the relocalization of OSBP was imaged by live-cell confocal laser scanning microscopy. Cells were imaged overnight. During the first 30 min, images were taken as fast as possible (∼1.5 min intervals), then intervals were stepwise increased to 30 min from 3.5 hr onward. Representative groups of cells are shown. The images are frames from Movie S1. (D) Quantification of the relative GFP-OSBP signal at the Golgi apparatus in five cells for each condition from (C). Error bars indicate SEM. Scale bar corresponds to 10 μm. See also Movie S1.

Figure 5

ITZ Binds OSBP and Inhibits Sterol and PI4P Transfer by OSBP (A–F) The effect of ITZ, antifungal azoles, or positive (25OH) or solvent controls (DMSO) on in vitro OSBP-mediated transfer of the fluorescent cholesterol analog DHE (A–C) or PI4P (D–F) was tested using liposomal assays depicted in (A) and (D). In both cases, initial exchange rates were determined in the presence of increasing concentrations of ITZ (B and E) or in the presence of 1 μM of the indicated drugs and then plotted in bar diagram (C and F). (G–J) The effect of ITZ on binding of an N-terminal OSBP fragment (amino acids 76–408; PH-FFAT) to ER-like (G and H) and Golgi-like (I and J) liposomes was examined by liposomal float-up assays as outlined in (G) and (I). Liposomal fractions were analyzed for binding of proteins by SDS-PAGE (H and J). (K and L) The effect of ITZ and control compounds on DHE (K) or PI4P (L) transfer by trypsinized OSBP was studied as with full-length OSBP (A–F). (M) The interaction of ITZ with GFP-tagged OSBP was investigated using MST. Data from three separate measurements were normalized and plotted, and a sigmoidal dose-response curve was fitted. Shown are mean values ± SEM. Statistical significance for the drug-treated conditions was calculated compared to the “no drug” control. See also Figure S7.

Figure 6

ITZ Affects OSBP Localization in Infected Cells (A) BGM cells were infected with CVB3 at MOI 10. At 4.5 hr p.i., cells were treated for 30 min with DMSO as vehicle control, 1 μM BF738753 (BF; a PI4KIIIβ inhibitor), or 10 μM ITZ. At 5 hr p.i., cells were fixed, processed for immunofluorescence with antibodies against OSBP and viral protein 3A, and imaged using confocal laser scanning microscopy. (B) Manders’ coefficients for overlap of 3A with OSBP were calculated for DMSO (12 cells), BF (7 cells) and ITZ (10 cells). Shown are means ± SEM. Asterisks indicate statistical significance compared to DMSO-treated controls. Scale bars correspond to 10 μm.

Figure 7

ITZ Inhibits PI4P and Cholesterol Shuttling in Infected Cells (A) BGM cells were infected with CVB3 at MOI 10. At 3 hr p.i., cells were treated for 1 hr with DMSO, 2 mM guanidine HCl (Gua), 1 μM BF738735 (BF), or 10 μM ITZ. At 4 hr p.i., cells were fixed, processed for immunofluorescence with antibodies against 3A and PI4P, imaged by wide-field microscopy, and deconvoluted. (B) PI4P intensity at 3A-positive structures was calculated for DMSO (11 cells), Gua (13 cells), BF (12 cells), and ITZ (11 cells). (C) HeLa R19 cells were infected with CVB3 at MOI 10. At 3 hr p.i., cells were treated for 1 hr with DMSO, 2 mM Gua, 1 μM BF, or 10 μM ITZ. At 4 hr p.i., cells were fixed, processed for immunofluorescence with an antibody against 3A and filipin to stain cholesterol, imaged by wide-field microscopy, and deconvoluted. (D) Pearson’s correlation coefficients for overlap of filipin and 3A were calculated for DMSO (17 cells), Gua (15 cells), BF (18 cells), and ITZ (19 cells). Shown are means ± SEM. Scale bars correspond to 10 μm.