Role of the lipid environment in NS5A structural rearrangements and drug interactions in hepatitis C

病毒蛋白在脂质环境中的功能研究尤其困难。丙型肝炎病毒（HCV）的非结构蛋白5A （NS5A）就是一个最突出的例子，尽管它是直接作用抗病毒药物（DAA）的关键靶点，但它仍然是最神秘的HCV蛋白之一。病毒复制与肝细胞的脂质代谢密切相关，胆固醇及其前体分子可以调节膜结合蛋白的生物活性。NS5A在内质网（ER；膜网）内新形成的膜上的病毒复制中起关键作用，并协助病毒在脂滴（ld）附近组装。针对NS5A抑制剂的耐药突变和相关的适应度代偿突变仅发生在NS5A-脂质界面，并可能在治疗失败后持续数年，对再治疗反应产生不利影响。尽管脂质在NS5A结构-功能关系中可能发挥关键作用，但解决脂质环境在NS5A结构重排中的作用的动态模型仍处于起步阶段。我们采用综合结构生物学方法，将计算技术与光学生物传感器技术等实验方法相结合，评估特定亚细胞脂质环境对NS5A构象和脂质引发的结构重排的影响。利用人工膜技术和合成生物学技术，模拟了经纯化的NS5A蛋白包埋的内质网和内质网膜脂的组成。我们将确定特异性的NS5A-脂质结合事件，研究相关的结构重排及其对NS5A自相互作用、寡聚和膜重塑的影响。我们的目的是了解膜脂与NS5A相互作用的机制细节，以探索RNA病毒生物学的基本生化原理。通过这种方式，我们也将表征NS5A抑制剂的作用机制和相关的病毒逃逸机制。

Functional studies of viral proteins in their lipid environment are particularly difficult. The non-structural protein 5A (NS5A) of the hepatitis C virus (HCV) is a most prominent example, that despite being a key target site of direct-acting antiviral agents (DAA), still is among the most enigmatic HCV proteins. Virus replication is closely tied to the lipid metabolism of liver cells with cholesterol and its precursor molecules known to modulate the biological activity of membrane-bound proteins. NS5A is critically involved in viral replication that takes place at newly formed membranes within the endoplasmic reticulum (ER; membranous web) and assists viral assembly in close vicinity of lipid droplets (LDs). Resistance mutations against NS5A inhibitors and related fitness-compensatory mutations exclusively occur at the NS5A-lipid interface, and potentially persist for years after treatment failure with adverse impact on retreatment responses. Despite the potential key role of lipids in the NS5A structure-function relationship, dynamic models to address the role of the lipid environment for structural rearrangements in NS5A are still in their infancy. We apply an integrated structural biology approach combining computational techniques with experimental approaches such as optical biosensor technology to assess the impact of specific subcellular lipid environments on NS5A conformation and lipid-triggered structural rearrangements. By using artificial membrane technology and synthetic biology, we mimic the ER and LD membrane lipid composition with purified NS5A protein embedded. We will identify specific NS5A-lipid binding events and study related structural rearrangements and their impact on NS5A self-interaction, oligomerization and membrane remodeling. Our aim is to understand mechanistic details in the interplay between membrane lipids and NS5A to explore fundamental biochemical principles in the biology of RNA viruses. In this way, we will also characterize the mechanisms of action of NS5A inhibitors and related viral escape mechanisms.