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

脂质环境在丙型肝炎 NS5A 结构重排和药物相互作用中的作用

• 批准号: 437572800
• 负责人: Privatdozent Dr. Christoph Welsch
• 金额: --
• 依托单位: Institut für Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/437572800?language=en
• 关键词: Role; lipid; environment; NS5A; structural

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.

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) 是最突出的例子，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已知可调节膜结合蛋白的生物活性。 NS5A 至关重要地参与在内质网（ER；膜网）内新形成的膜上发生的病毒复制，并协助病毒在脂滴 (LD) 附近组装。针对 NS5A 抑制剂的耐药性突变和相关的适应性补偿突变仅发生在 NS5A-脂质界面上，并且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-功能关系中具有潜在的关键作用，但解决脂质环境对 NS5A 结构重排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，以评估特定亚细胞脂质环境对 NS5A 构象和脂质触发的结构重排的影响。通过使用人工膜技术和合成生物学，我们模拟了内嵌有纯化的 NS5A 蛋白的 ER 和 LD 膜脂组成。我们将识别特定的 NS5A-脂质结合事件，并研究相关的结构重排及其对 NS5A 自身相互作用、寡聚化和膜重塑的影响。我们的目标是了解膜脂和 NS5A 之间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，我们还将表征 NS5A 抑制剂的作用机制和相关的病毒逃逸机制。