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