Mechanistic function of HCV NS5A targeted by the potent inhibitors

高效NS5A抑制剂(NS5A-I)的开发对丙型肝炎病毒(HCV)治疗的成功起到了关键作用，其治愈率接近100%。值得注意的是，NS5A-I的分子作用机制和NS5A的确切分子功能都不清楚。这一应用的目标是通过检验我们的中心假设来解决丙型肝炎病毒领域的这一主要缺口，该假设认为NS5A-I阻止NS5A高阶寡聚体(h-寡聚体)，这些低聚体在丙型肝炎病毒复制过程中双膜泡(DMV)的形成中具有直接的膜重塑作用。我们的初步研究表明，NS5A-I可以破坏由NS5A-N-末端结构域形成的高阶低聚物(NS5A-NTD)。此外，通过使用创新的共聚焦时移成像系统，我们获得了NS5A-I可以抑制NS5A-NTD介导的膜重构的证据。有了这些支持我们假设的初步数据，我们现在将NS5A的h-寡聚和膜重构定义为NS5A-I的直接靶点，在特定的目的1.特定的目的2将阐明NS5A的不同结构域在NS5A的h-寡聚体依赖的体外膜重构和表达NS5A的细胞中DMV形成中的作用。在特定的目标3中，我们将阐明胆固醇在NS5A介导的膜重塑中的作用。该项目的结果可能会对产生膜保护复制间隔的其他病毒以及针对NS5A类靶点的合理药物设计领域产生广泛影响。

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.