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.
Despite the availability of effective antivirals, ~71 million people are still infected with chronic hepatitis C virus (HCV) that causes 399,000 liver disease-related deaths per year. This project aims to reveal, at last, the molecular mechanism of action of the key antiviral component of successful HCV therapy, which targets HCV NS5A. This project could have a broad impact on rational drug design for NS5A-like viral (or cellular) targets that induce membrane remodeling.
