The development of highly potent NS5A inhibitors (NS5A-i) played a key role in the successful development of hepatitis C virus (HCV) therapy with a near 100% cure rate. Remarkably, neither the molecular mechanism of action of NS5A-i nor the exact molecular functions of NS5A specifically targeted by NS5A-i are known. The goal of this application is to address this major gap in the HCV field by testing our central hypothesis that NS5A-i block NS5A high-order oligomers (h-oligomers), which have a direct role in membrane remodeling for the double membrane vesicles (DMV) formation during HCV replication. Our preliminary study showed that NS5A-i could disrupt the high-order oligomers formed by NS5A-N-terminal domain (NS5A-NTD). In addition, by using an innovative confocal time-lapse imaging system, we obtained evidence that NS5A-i could inhibit NS5A- NTD-mediated membrane remodeling. Armed with these preliminary data supporting our hypothesis, we will now define NS5A h-oligomerization and membrane remodeling as a direct target of NS5A-i by using a novel in vitro model system in Specific Aim 1. Specific Aim 2 will elucidate the role of different domains of NS5A in NS5A h-oligomer-dependent membrane remodeling in vitro and DMV formation in NS5A expressing cells. In Specific Aim 3, we will elucidate the role of cholesterol in NS5A-mediated membrane remodeling. The outcome of this project could have a broad impact on the field for other viruses that generate membrane-protected replication compartments, as well as rational drug design for NS5A-like targets.