LAUSR

ABSTRACT The genus Enterovirus (e.g. poliovirus, coxsackievirus, rhinovirus) of the Picornaviridae family of positive‐strand RNA viruses includes many important pathogens linked to a range of acute and chronic diseases for which no approved antiviral therapy is available. Targeting a step in the life cycle that is highly conserved provides an attractive strategy for developing broad‐range inhibitors of enterovirus infection. A step that is currently explored as a target for the development of antivirals is the formation of replication organelles, which support replication of the viral genome. To build replication organelles, enteroviruses rewire cellular machinery and hijack lipid homeostasis pathways. For example, enteroviruses exploit the PI4KIII&bgr;‐PI4P‐OSBP pathway to direct cholesterol to replication organelles. Here, we uncover that TTP‐8307, a known enterovirus replication inhibitor, acts through the PI4KIII&bgr;‐PI4P‐OSBP pathway by directly inhibiting OSBP activity. However, despite a shared mechanism of TTP‐8307 with established OSBP inhibitors (itraconazole and OSW‐1), we identify a number of notable differences between these compounds. The antiviral activity of TTP‐8307 extends to other viruses that require OSBP, namely the picornavirus encephalomyocarditis virus and the flavivirus hepatitis C virus. HIGHLIGHTSTTP‐8307 inhibits enterovirus replication.Mutations in viral protein 3A that provide resistance to PI4KIII&bgr; and OSBP inhibitors also provide resistance to TTP‐8307.TTP‐8307 does not inhibit PI4KIII&bgr; activity.TTP‐8307 inhibits OSBP‐mediated lipid shuttling.OSBP inhibitors TTP‐8307, OSW‐1 and itraconazole have different mechanisms of inhibition.