Chukkapalli Vineela, Berger Kristi L, Kelly Sean M, Thomas Meryl, Deiters Alexander, Randall Glenn
Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA.
Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Virology. 2015 Feb;476:168-179. doi: 10.1016/j.virol.2014.12.018. Epub 2014 Dec 26.
Combinations of direct-acting antivirals (DAAs) against the hepatitis C virus (HCV) have the potential to revolutionize the HCV therapeutic regime. An integral component of DAA combination therapies is HCV NS5A inhibitors. It has previously been proposed that NS5A DAAs inhibit two functions of NS5A: RNA replication and virion assembly. In this study, we characterize the impact of a prototype NS5A DAA, daclatasvir (DCV), on HCV replication compartment formation. DCV impaired HCV replicase localization and NS5A motility. In order to characterize the mechanism behind altered HCV replicase localization, we examined the impact of DCV on the interaction of NS5A with its essential cellular cofactor, phosphatidylinositol-4-kinase III α (PI4KA). We observed that DCV does not inhibit PI4KA directly, nor does it impair early events of the NS5A-PI4KA interaction that can occur when NS5A is expressed alone. NS5A functions that are unaffected by DCV include PI4KA binding, as determined by co-immunoprecipitation, and a basal accumulation of the PI4KA product, PI4P. However, DCV impairs late steps in PI4KA activation that requires NS5A expressed in the context of the HCV polyprotein. These NS5A functions include hyper-stimulation of PI4P levels and appropriate replication compartment formation. The data are most consistent with a model wherein DCV inhibits conformational changes in the NS5A protein or protein complex formations that occur in the context of HCV polyprotein expression and stimulate PI4P hyper-accumulation and replication compartment formation.
针对丙型肝炎病毒（HCV）的直接作用抗病毒药物（DAA）组合有潜力彻底改变HCV治疗方案。DAA联合疗法的一个重要组成部分是HCV NS5A抑制剂。此前有人提出，NS5A DAA可抑制NS5A的两种功能：RNA复制和病毒粒子组装。在本研究中，我们描述了一种原型NS5A DAA——达卡他韦（DCV）对HCV复制区室形成的影响。DCV损害了HCV复制酶的定位和NS5A的运动性。为了描述HCV复制酶定位改变背后的机制，我们研究了DCV对NS5A与其必需的细胞辅因子磷脂酰肌醇-4-激酶IIIα（PI4KA）相互作用的影响。我们观察到，DCV不会直接抑制PI4KA，也不会损害单独表达NS5A时可能发生的NS5A-PI4KA相互作用的早期事件。不受DCV影响的NS5A功能包括通过免疫共沉淀确定的PI4KA结合以及PI4KA产物PI4P的基础积累。然而，DCV会损害PI4KA激活的后期步骤，而这需要在HCV多聚蛋白背景下表达的NS5A。这些NS5A功能包括PI4P水平的过度刺激和适当的复制区室形成。这些数据与一个模型最为一致，即DCV抑制在HCV多聚蛋白表达背景下发生的NS5A蛋白构象变化或蛋白复合物形成，并刺激PI4P过度积累和复制区室形成。
