1Key Laboratory of Medical Molecular Virology and Department of Medical Microbiology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, PR China.
2Department of Pathogen Diagnosis and Biosafety, Shanghai Public Health Clinical Center, Fudan University, Shanghai, PR China.
J Gen Virol. 2019 Jan;100(1):69-83. doi: 10.1099/jgv.0.001180. Epub 2018 Dec 5.
Daclatasvir (DCV) is a highly potent direct-acting antiviral that targets the non-structural protein 5A (NS5A) of hepatitis C virus (HCV) and has been used with great clinical success. Previous studies have demonstrated its impact on viral replication complex assembly. However, the precise mechanisms by which DCV impairs the replication complex assembly remains elusive. In this study, by using HCV subgenomic replicons and a viral replicase assembly surrogate system in which the HCV NS3-5B polyprotein is expressed to mimic the viral replicase assembly, we assessed the impact of DCV on the aggregation and tertiary structure of NS5A, the protein-protein interactions within the viral replicase and the quaternary structure of the viral replicase. We found that DCV did not affect aggregation and tertiary structure of NS5A. DCV induced a quaternary structural change of the viral replicase, as evidenced by selective increase of NS4B's sensitivity to proteinase K digestion. Mechanically, DCV impaired the NS4B-involved protein-protein interactions within the viral replicase. These phenotypes were consistent with the phenotypes of several reported NS4B mutants that abolish the viral replicase assembly. The DCV-resistant mutant Y93H was refractory to the DCV-induced reduction of the NS4B-involved protein interactions and the quaternary structural change of the viral replicase. In addition, Y93H reduced NS4B-involved protein-protein interactions within the viral replicase and attenuated viral replication. We propose that DCV may induce a positional change of NS5A, which allosterically affects protein interactions within the replicase components and disrupts replicase assembly.
达卡他韦（DCV）是一种高效的直接作用抗病毒药物，靶向丙型肝炎病毒（HCV）的非结构蛋白 5A（NS5A），并已在临床应用中取得了巨大成功。先前的研究表明它对病毒复制复合物组装有影响。然而，DCV 损害复制复合物组装的确切机制仍不清楚。在这项研究中，我们使用 HCV 亚基因组复制子和病毒复制酶组装替代系统，其中 HCV NS3-5B 多蛋白表达来模拟病毒复制酶组装，评估了 DCV 对 NS5A 聚集和三级结构、病毒复制酶内的蛋白-蛋白相互作用以及病毒复制酶的四级结构的影响。我们发现 DCV 不影响 NS5A 的聚集和三级结构。DCV 诱导病毒复制酶的四级结构发生变化，这表现在 NS4B 对蛋白水解酶的敏感性选择性增加。从机制上讲，DCV 损害了病毒复制酶内涉及 NS4B 的蛋白-蛋白相互作用。这些表型与几种报道的 NS4B 突变体一致，这些突变体破坏了病毒复制酶的组装。DCV 耐药突变体 Y93H 对 DCV 诱导的减少 NS4B 涉及的蛋白相互作用和病毒复制酶的四级结构变化没有反应。此外，Y93H 减少了病毒复制酶内涉及 NS4B 的蛋白-蛋白相互作用并减弱了病毒复制。我们提出，DCV 可能诱导 NS5A 的位置变化，这种变构作用影响复制酶成分内的蛋白相互作用并破坏复制酶组装。
