Physical Chemistry, ETH Zurich, 8093, Zurich, Switzerland.
Molecular Microbiology and Structural Biochemistry, Labex Ecofect, UMR 5086 CNRS, Université de Lyon 1, 7 passage du Vercors, 69367, Lyon, France.
Angew Chem Int Ed Engl. 2021 Mar 1;60(10):5339-5347. doi: 10.1002/anie.202013296. Epub 2021 Jan 18.
The Hepatitis C virus nonstructural protein 5A (NS5A) is a membrane-associated protein involved in multiple steps of the viral life cycle. Direct-acting antivirals (DAAs) targeting NS5A are a cornerstone of antiviral therapy, but the mode-of-action of these drugs is poorly understood. This is due to the lack of information on the membrane-bound NS5A structure. Herein, we present the structural model of an NS5A AH-linker-D1 protein reconstituted as proteoliposomes. We use highly sensitive proton-detected solid-state NMR methods suitable to study samples generated through synthetic biology approaches. Spectra analyses disclose that both the AH membrane anchor and the linker are highly flexible. Paramagnetic relaxation enhancements (PRE) reveal that the dimer organization in lipids requires a new type of NS5A self-interaction not reflected in previous crystal structures. In conclusion, we provide the first characterization of NS5A AH-linker-D1 in a lipidic environment shedding light onto the mode-of-action of clinically used NS5A inhibitors.
丙型肝炎病毒非结构蛋白 5A（NS5A）是一种与膜相关的蛋白，参与病毒生命周期的多个步骤。直接作用抗病毒药物（DAAs）是抗病毒治疗的基石，但这些药物的作用机制尚不清楚。这是由于缺乏关于膜结合 NS5A 结构的信息。在此，我们提出了一种 NS5A AH-连接子-D1 蛋白作为脂蛋白重建的结构模型。我们使用高灵敏度的质子检测固态 NMR 方法，适用于通过合成生物学方法生成的样品。谱分析表明，AH 膜锚和连接子都具有高度的灵活性。顺磁弛豫增强（PRE）揭示了脂质中二聚体的组织需要一种新的 NS5A 自身相互作用，这在以前的晶体结构中没有反映。总之，我们首次在脂质环境中对 NS5A AH-连接子-D1 进行了表征，为临床使用的 NS5A 抑制剂的作用机制提供了线索。
