Mechanistic function of HCV NS5A targeted by the potent inhibitors

高效NS5A抑制剂（NS5A-i）的开发在丙型肝炎病毒（HCV）治疗的成功开发中发挥了关键作用，其治愈率接近100%。值得注意的是，NS5A-i的分子作用机制和NS5A-i特异性靶向NS5A的确切分子功能尚不清楚。本应用程序的目的是通过验证我们的中心假设，即NS5A-i阻断NS5A高阶低聚物（h-寡聚物），解决HCV领域的这一主要空白，NS5A-i在HCV复制过程中双膜囊泡（DMV）形成的膜重塑中起直接作用。我们的初步研究表明，NS5A-i可以破坏ns5a - n末端结构域（NS5A-NTD）形成的高阶低聚物。此外，通过创新的共聚焦延时成像系统，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，我们现在将通过在Specific Aim 1中使用一种新的体外模型系统，将NS5A h-寡聚化和膜重塑定义为NS5A-i的直接靶点。特异性目的2将阐明NS5A不同结构域在体外NS5A h-寡聚物依赖性膜重构和表达NS5A细胞DMV形成中的作用。在Specific Aim 3中，我们将阐明胆固醇在ns5a介导的膜重构中的作用。这个项目的结果可能会对其他产生膜保护复制区的病毒领域产生广泛的影响，以及对ns5a样靶标的合理药物设计。

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.