Mechanistic function of HCV NS5A targeted by the potent inhibitors

高效NS 5A抑制剂（NS 5A-i）的开发在成功开发丙型肝炎病毒（HCV）治疗中发挥了关键作用，治愈率接近100%。值得注意的是，NS 5A-i的分子作用机制和NS 5A-i特异性靶向的NS 5A的确切分子功能均未知。本申请的目标是通过测试我们的中心假设，即NS 5A-i阻断NS 5A高级寡聚体（h-寡聚体），来解决HCV领域的这一主要空白，所述NS 5A高级寡聚体在HCV复制期间对双膜囊泡（DMV）形成的膜重塑中具有直接作用。我们的初步研究表明，NS 5A-i可以破坏由NS 5A-N-末端结构域（NS 5A-NTD）形成的高级寡聚体。此外，通过使用创新的共聚焦延时成像系统，我们获得了NS 5A-i可以抑制NS 5A-NTD介导的膜重塑的证据。有了这些支持我们假设的初步数据，我们现在将通过使用特异性目的1中的新型体外模型系统将NS 5A h-寡聚化和膜重塑定义为NS 5A-i的直接靶标。具体目标2将阐明NS 5A的不同结构域在体外NS 5A h-寡聚体依赖性膜重塑和NS 5A表达细胞中DMV形成中的作用。在具体目标3中，我们将阐明胆固醇在NS 5A介导的膜重塑中的作用。该项目的结果可能对其他产生膜保护复制区室的病毒领域产生广泛影响，以及NS 5A样靶点的合理药物设计。

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.