SUMMARY Itraconazole (ITZ) is a well-known antifungal agent that also has anti-cancer activity. In this study, we identified 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 novel 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.

Viruses are thought to spread across susceptible cells through an iterative process of infection, replication, and release, so that the rate of spread is limited by replication kinetics. Here, we show that vaccinia virus spreads across one cell every 75 minutes, fourfold faster than its replication cycle would permit. To explain this phenomenon, we found that newly infected cells express two surface proteins that mark cells as infected and, via exploitation of cellular machinery, induce the repulsion of superinfecting virions away toward uninfected cells. Mechanistically, early expression of proteins A33 and A36 was critical for virion repulsion and rapid spread, and cells expressing these proteins repelled exogenous virions rapidly. Additional spreading mechanisms may exist for other viruses that also spread faster than predicted by replication kinetics.Mechanisms enhancing the cell-to-cell spread of intracellular pathogens are important for virulence and are targets for development of antimicrobial therapeutics. Vaccinia virus (VACV) is a poxvirus and is the live vaccine used to eradicate smallpox (1). VACV replication is unusual in that it produces both single-and double-enveloped virions (2, 3). The single-enveloped virions, called intracellular mature virus (IMV), remain intracellular until cell lysis and spread slowly from cell to cell. In contrast, the double-enveloped virions, called cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV), are released rapidly and mediate efficient cell-to-cell spread and long-range dissemination (3, 4). VACV spreading mechanisms include virus-induced cell motility (5) and the formation of actin projections (6-8) that propel VACV particles toward other cells late during infection (9). However, we wondered whether either mechanism could explain how VACV Western Reserve (WR) spreads rapidly to form a plaque of diameter 2.90 ± 0.07 mm (SEM, nine experiments, n = 11 to 12 plaques) in 3 days (Fig. 1A). The distance between nuclei of adjacent BSC-1 cells was 37.26 ± 1.02 μm (SEM, n = 25 single cells and the 5 to 8 cells in Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts contact with it), so that VACV was spreading across each cell in <2 hours. To study this further, live video microscopy was used to measure the spread of VACV-induced cytopathic effect after infection with VACV WR (Fig. 1, B and C) or VACV expressing enhanced green fluorescent protein (EGFP) fused to core protein A5 that is expressed late during infection (vEGFPA5L) (Fig. 1, D to F, and movies S1 to S5) (10, 11). A linear increase in plaque size with time was observed (Fig. 1G), and the mean rate of spread was 32.36 ± 0.74 μm/hour (SEM, n = 9 plaques) for VACV WR. Knowing the distance between nuclei of adjacent cells, this indicated that VACV crossed one cell every 1.2 hours. This rate of spread is inconsistent with VACV replication kinetics, in which new virions are formed only 5 to 6 hours after infection (12), or virus-induced cell motility, in which ...

RNA viruses can rapidly mutate and acquire resistance to drugs that directly target viral enzymes, which poses serious problems in a clinical context. Therefore, there is a growing interest in the development of antiviral drugs that target host factors critical for viral replication, since they are unlikely to mutate in response to therapy. We recently demonstrated that phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) and its product phosphatidylinositol-4-phosphate (PI4P) are essential for replication of enteroviruses, a group of medically important RNA viruses including poliovirus (PV), coxsackievirus, rhinovirus, and enterovirus 71. Here, we show that enviroxime and GW5074 decreased PI4P levels at the Golgi complex by directly inhibiting PI4KIIIβ. Coxsackievirus mutants resistant to these inhibitors harbor single point mutations in the non-structural protein 3A. These 3A mutations did not confer compound-resistance by restoring the activity of PI4KIIIβ in the presence of the compounds. Instead, replication of the mutant viruses no longer depended on PI4KIIIβ, since their replication was insensitive to siRNA-mediated depletion of PI4KIIIβ. The mutant viruses also did not rely on other isoforms of PI4K. Consistently, no high level of PI4P could be detected at the replication sites induced by the mutant viruses in the presence of the compounds. Collectively, these findings indicate that through specific single point mutations in 3A, CVB3 can bypass an essential host factor and lipid for its propagation, which is a new example of RNA viruses acquiring resistance against antiviral compounds, even when they directly target host factors.