Background & Aims Positive-sense RNA viruses remodel intracellular membranes to generate specialized membrane compartments for viral replication. Several RNA viruses, including poliovirus and hepatitis C virus (HCV), require phosphatidylinositol (PI) 4-kinases for their replication. However, it is not known how PI 4-kinases and their product, PI(4)P, facilitate host membrane reorganization and viral replication. In addition, although the HCV replication compartment, known as the membranous web, is believed to be cholesterol enriched, the mechanisms by which this occurs have not been elucidated. We aimed to identify and characterize a PI 4-kinase effector in HCV replication. Methods We used a combination of microscopic and biochemical methods to study HCV replication, web morphology, the distribution of intracellular protein and PI(4)P, along with cholesterol trafficking in HCV-infected cells. PI 4-kinase and oxysterol-binding protein (OSBP) were inhibited using RNA interference or small molecules in cells expressing a full-length genotype 1b replicon or infected with the JFH-1 strain of HCV. Results OSBP was required for HCV replication and membranous web integrity. OSBP was recruited to membranous webs in a PI 4-kinase-dependent manner, and both these factors were found to regulate cholesterol trafficking to the web. We also found OSBP to be required for poliovirus infection but dispensable for dengue virus. Conclusions OSBP is a PI 4-kinase effector in HCV infection, and contributes to the integrity and cholesterol enrichment of the membranous web. OSBP might also be a PI 4-kinase effector in poliovirus infection and could be involved in replication of other viruses that require PI 4-kinases
