Reghellin V, Donnici L, Fenu S, Berno V, Calabrese V, Pagani M, Abrignani S, Peri F, De Francesco R, Neddermann P
Istituto Nazionale Genetica Molecolare (INGM) Romeo ed Enrica Invernizzi, Milan, Italy.
Department of Biotechnology and Biosciences, University of Milano Bicocca, Milan, Italy.
Antimicrob Agents Chemother. 2014 Dec;58(12):7128-40. doi: 10.1128/AAC.03293-14. Epub 2014 Sep 15.
The hepatitis C virus (HCV) nonstructural (NS) protein 5A is a multifunctional protein that plays a central role in viral replication and assembly. Antiviral agents directly targeting NS5A are currently in clinical development. Although the elucidation of the mechanism of action (MOA) of NS5A inhibitors has been the focus of intensive research, a detailed understanding of how these agents exert their antiviral effect is still lacking. In this study, we observed that the downregulation of NS5A hyperphosphorylation is associated with the actions of NS5A inhibitors belonging to different chemotypes. NS5A is known to recruit the lipid kinase phosphatidylinositol 4-kinase IIIα (PI4KIIIα) to the HCV-induced membranous web in order to generate phosphatidylinositol 4-phosphate (PI4P) at the sites of replication. We demonstrate that treatment with NS5A inhibitors leads to an impairment in the NS5A-PI4KIIIα complex formation that is paralleled by a significant reduction in PI4P and cholesterol levels within the endomembrane structures of HCV-replicating cells. A similar decrease in PI4P and cholesterol levels was also obtained upon treatment with a PI4KIIIα-targeting inhibitor. In addition, both the NS5A and PI4KIIIα classes of inhibitors induced similar subcellular relocalization of the NS5A protein, causing the formation of large cytoplasmic NS5A-containing clusters previously reported to be one of the hallmarks of inhibition of the action of PI4KIIIα. Because of the similarities between the effects induced by treatment with PI4KIIIα or NS5A inhibitors and the observation that agents targeting NS5A impair NS5A-PI4KIIIα complex formation, we speculate that NS5A inhibitors act by interfering with the function of the NS5A-PI4KIIIα complex.
丙型肝炎病毒（HCV）非结构（NS）蛋白5A是一种多功能蛋白，在病毒复制和组装过程中发挥核心作用。目前，直接靶向NS5A的抗病毒药物正处于临床开发阶段。尽管对NS5A抑制剂作用机制（MOA）的阐明一直是深入研究的重点，但仍缺乏对这些药物如何发挥抗病毒作用的详细了解。在本研究中，我们观察到NS5A过度磷酸化的下调与不同化学类型的NS5A抑制剂的作用相关。已知NS5A会将脂质激酶磷脂酰肌醇4激酶IIIα（PI4KIIIα）募集到HCV诱导的膜网中，以便在复制位点生成磷脂酰肌醇4磷酸（PI4P）。我们证明，用NS5A抑制剂处理会导致NS5A-PI4KIIIα复合物形成受损，同时HCV复制细胞内膜结构中的PI4P和胆固醇水平显著降低。用靶向PI4KIIIα的抑制剂处理后，PI4P和胆固醇水平也出现了类似的下降。此外，NS5A和PI4KIIIα两类抑制剂均诱导了NS5A蛋白类似的亚细胞重新定位，导致形成大量含NS5A的细胞质簇，此前报道这是抑制PI4KIIIα作用的标志之一。由于用PI4KIIIα或NS5A抑制剂处理所诱导的效应之间存在相似性，以及靶向NS5A的药物会损害NS5A-PI4KIIIα复合物形成这一观察结果，我们推测NS5A抑制剂通过干扰NS5A-PI4KIIIα复合物的功能发挥作用。
