Limits...
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.

Show MeSH

Related in: MedlinePlus

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, 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 10hrs 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, infected with CVB3, EV71, or HRV2 at MOI 1 and virus titers at 10 hr p.i. were determined by endpoint titration. Knockdown efficiency was determined by Q-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 EGFP, 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 10µm. See also Figures S5 and S6.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4383725&req=5

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, 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 10hrs 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, infected with CVB3, EV71, or HRV2 at MOI 1 and virus titers at 10 hr p.i. were determined by endpoint titration. Knockdown efficiency was determined by Q-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 EGFP, 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 10µm. See also Figures S5 and S6.

Mentions: Having ruled out PI4KIIIβ as a target of ITZ, we next turned to signaling steps downstream of PI4P, i.e., proteins that bind to PI4P lipids. To assess whether any of the known PI4P-binding proteins could be a target of ITZ, we performed a target identification by siRNA sensitization (TISS) assay (Arita et al., 2010). TISS encompasses siRNA knockdown of candidate target proteins to potentiate the biological effect of a low concentration of a compound. Among a number of PI4P-binding, Golgi-localized proteins, knockdown of OSBP, but not any of the other PH domain-containing proteins, enhanced the inhibitory effect of a low concentration (1.25 µM) of ITZ on PV replication (Figure 3A), implying OSBP as a possible antiviral target of ITZ. We further assessed this possibility by several experiments. First, the OSBP antagonist OSW-1 (Burgett et al., 2011) potently inhibited CVB3 replication (Figure 3B), confirming that pharmacological targeting of OSBP can inhibit enterovirus replication. As for ITZ, the 3A[H57Y] mutation in CVB3 provided resistance against OSW-1 (Figure 3B). Akin to ITZ, OSW-1 inhibited all enteroviruses tested as well as EMCV, but not ERAV (data not shown). Importantly, OSW-1 did not affect endolysosomal cholesterol distribution (Figure 3C), supporting our previous conclusion that this effect unlikely explains the antiviral effect of ITZ. Second, similar as for PV (Wang et al., 2014) siRNA knockdown of OSBP inhibited replication of EV71 and HRV2 (Figure 3D). CVB3 replication was also inhibited by OSBP knockdown but this difference was not statistically significant, in line with the lower sensitivity of CVB3 than EV71 to ITZ (Figure 1A). Third, overexpression of OSBP restored replication of CVB3 and EV71 in the presence of both ITZ or OSW-1 (Figure 3E), confirming that inhibition of viral replication by ITZ and OSW-1 is mediated through OSBP. Overexpression of PI4KIIIβ failed to rescue replication, nor did OSBP overexpression provide rescue against PI4KIIIβ inhibitors (data not shown), indicating the specificity of the experimental setup.


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 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, 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 10hrs 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, infected with CVB3, EV71, or HRV2 at MOI 1 and virus titers at 10 hr p.i. were determined by endpoint titration. Knockdown efficiency was determined by Q-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 EGFP, 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 10µm. See also Figures S5 and S6.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383725&req=5

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, 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 10hrs 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, infected with CVB3, EV71, or HRV2 at MOI 1 and virus titers at 10 hr p.i. were determined by endpoint titration. Knockdown efficiency was determined by Q-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 EGFP, 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 10µm. See also Figures S5 and S6.
Mentions: Having ruled out PI4KIIIβ as a target of ITZ, we next turned to signaling steps downstream of PI4P, i.e., proteins that bind to PI4P lipids. To assess whether any of the known PI4P-binding proteins could be a target of ITZ, we performed a target identification by siRNA sensitization (TISS) assay (Arita et al., 2010). TISS encompasses siRNA knockdown of candidate target proteins to potentiate the biological effect of a low concentration of a compound. Among a number of PI4P-binding, Golgi-localized proteins, knockdown of OSBP, but not any of the other PH domain-containing proteins, enhanced the inhibitory effect of a low concentration (1.25 µM) of ITZ on PV replication (Figure 3A), implying OSBP as a possible antiviral target of ITZ. We further assessed this possibility by several experiments. First, the OSBP antagonist OSW-1 (Burgett et al., 2011) potently inhibited CVB3 replication (Figure 3B), confirming that pharmacological targeting of OSBP can inhibit enterovirus replication. As for ITZ, the 3A[H57Y] mutation in CVB3 provided resistance against OSW-1 (Figure 3B). Akin to ITZ, OSW-1 inhibited all enteroviruses tested as well as EMCV, but not ERAV (data not shown). Importantly, OSW-1 did not affect endolysosomal cholesterol distribution (Figure 3C), supporting our previous conclusion that this effect unlikely explains the antiviral effect of ITZ. Second, similar as for PV (Wang et al., 2014) siRNA knockdown of OSBP inhibited replication of EV71 and HRV2 (Figure 3D). CVB3 replication was also inhibited by OSBP knockdown but this difference was not statistically significant, in line with the lower sensitivity of CVB3 than EV71 to ITZ (Figure 1A). Third, overexpression of OSBP restored replication of CVB3 and EV71 in the presence of both ITZ or OSW-1 (Figure 3E), confirming that inhibition of viral replication by ITZ and OSW-1 is mediated through OSBP. Overexpression of PI4KIIIβ failed to rescue replication, nor did OSBP overexpression provide rescue against PI4KIIIβ inhibitors (data not shown), indicating the specificity of the experimental setup.

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.

Show MeSH
Related in: MedlinePlus