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Itraconazole inhibits enterovirus replication by targeting the oxysterol-binding protein.

Strating JR, van der Linden L, Albulescu L, Bigay J, Arita M, Delang L, Leyssen P, van der Schaar HM, Lanke KH, Thibaut HJ, Ulferts R, Drin G, Schlinck N, Wubbolts RW, Sever N, Head SA, Liu JO, Beachy PA, De Matteis MA, Shair MD, Olkkonen VM, Neyts J, van Kuppeveld FJ - Cell Rep (2015)

Bottom Line: Itraconazole (ITZ) is a well-known antifungal agent that also has anticancer activity.We demonstrate that ITZ inhibits viral RNA replication by targeting oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4).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.

View Article: PubMed Central - PubMed

Affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584CL Utrecht, the Netherlands; Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, 6525GA Nijmegen, the Netherlands.

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ITZ does not inhibit virus replication through known targets or PI4KIIIβ, although CVB3 with mutations in the non-structural viral protein 3A are cross-resistant to ITZ and PI4KIIIβ inhibitors(A, B, D) HeLa R19 (A) or BGM (B, D) cells were infected with RLuc-CVB3 at MOI 0.1, treated with 10 µM ITZ, DMSO, or 10 µM antifungal azoles (A), Hedgehog pathway antagonists (100nM 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, 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 three days. (G–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, B) or of mutant virus compared to wt. Scale bars correspond to 10µm. See also Figures S3 and S4.
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Figure 2: ITZ does not inhibit virus replication through known targets or PI4KIIIβ, although CVB3 with mutations in the non-structural viral protein 3A are cross-resistant to ITZ and PI4KIIIβ inhibitors(A, B, D) HeLa R19 (A) or BGM (B, D) cells were infected with RLuc-CVB3 at MOI 0.1, treated with 10 µM ITZ, DMSO, or 10 µM antifungal azoles (A), Hedgehog pathway antagonists (100nM 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, 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 three days. (G–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, B) or of mutant virus compared to wt. Scale bars correspond to 10µm. See also Figures S3 and S4.

Mentions: ITZ is widely used as an antifungal drug that inhibits the fungal enzyme CYP51. ITZ has also been shown to have some inhibitory activity towards the human CYP51 (hCYP51) and the related cytochrome P450 CYP3A4. In addition to ITZ, other azole family antifungal drugs, including posaconazole, ketoconazole, fluconazole and voriconazole (Figure S1), also inhibit hCYP51 and CYP3A4 with slightly lower or similar potency as ITZ (Warrilow et al., 2013; Zhang et al., 2012). We tested whether these drugs exert antiviral activity using recombinant viruses RLuc-CVB3 and RLuc-EMCV, which carry the Renilla luciferase gene upstream of the coding region. At 10 µM, only posaconazole inhibited replication of RLuc-CVB3 and RLuc-EMCV. The remaining azoles did not display any antiviral activity at concentrations up to 100 µM (Figures 2A and S3A–C). Similar results were obtained in a multi-cycle CPE-reduction assay (not shown). These results ruled out the possibility that inhibition of hCYP51 or CYP3A4 underlies the antiviral activity of ITZ and its structurally related analog posaconazole.


Itraconazole inhibits enterovirus replication by targeting the oxysterol-binding protein.

Strating JR, van der Linden L, Albulescu L, Bigay J, Arita M, Delang L, Leyssen P, van der Schaar HM, Lanke KH, Thibaut HJ, Ulferts R, Drin G, Schlinck N, Wubbolts RW, Sever N, Head SA, Liu JO, Beachy PA, De Matteis MA, Shair MD, Olkkonen VM, Neyts J, van Kuppeveld FJ - Cell Rep (2015)

ITZ does not inhibit virus replication through known targets or PI4KIIIβ, although CVB3 with mutations in the non-structural viral protein 3A are cross-resistant to ITZ and PI4KIIIβ inhibitors(A, B, D) HeLa R19 (A) or BGM (B, D) cells were infected with RLuc-CVB3 at MOI 0.1, treated with 10 µM ITZ, DMSO, or 10 µM antifungal azoles (A), Hedgehog pathway antagonists (100nM 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, 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 three days. (G–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, B) or of mutant virus compared to wt. Scale bars correspond to 10µm. See also Figures S3 and S4.
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Figure 2: ITZ does not inhibit virus replication through known targets or PI4KIIIβ, although CVB3 with mutations in the non-structural viral protein 3A are cross-resistant to ITZ and PI4KIIIβ inhibitors(A, B, D) HeLa R19 (A) or BGM (B, D) cells were infected with RLuc-CVB3 at MOI 0.1, treated with 10 µM ITZ, DMSO, or 10 µM antifungal azoles (A), Hedgehog pathway antagonists (100nM 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, 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 three days. (G–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, B) or of mutant virus compared to wt. Scale bars correspond to 10µm. See also Figures S3 and S4.
Mentions: ITZ is widely used as an antifungal drug that inhibits the fungal enzyme CYP51. ITZ has also been shown to have some inhibitory activity towards the human CYP51 (hCYP51) and the related cytochrome P450 CYP3A4. In addition to ITZ, other azole family antifungal drugs, including posaconazole, ketoconazole, fluconazole and voriconazole (Figure S1), also inhibit hCYP51 and CYP3A4 with slightly lower or similar potency as ITZ (Warrilow et al., 2013; Zhang et al., 2012). We tested whether these drugs exert antiviral activity using recombinant viruses RLuc-CVB3 and RLuc-EMCV, which carry the Renilla luciferase gene upstream of the coding region. At 10 µM, only posaconazole inhibited replication of RLuc-CVB3 and RLuc-EMCV. The remaining azoles did not display any antiviral activity at concentrations up to 100 µM (Figures 2A and S3A–C). Similar results were obtained in a multi-cycle CPE-reduction assay (not shown). These results ruled out the possibility that inhibition of hCYP51 or CYP3A4 underlies the antiviral activity of ITZ and its structurally related analog posaconazole.

Bottom Line: Itraconazole (ITZ) is a well-known antifungal agent that also has anticancer activity.We demonstrate that ITZ inhibits viral RNA replication by targeting oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4).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.

View Article: PubMed Central - PubMed

Affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584CL Utrecht, the Netherlands; Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, 6525GA Nijmegen, the Netherlands.

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Related in: MedlinePlus