TIP47 plays a crucial role in the life cycle of hepatitis C virus
TIP47 plays a crucial role in the life cycle of hepatitis C virus
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DOI：
10.1016/j.jhep.2013.01.022
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发表时间：
2013-06-01
影响因子：
25.7
通讯作者：
Hildt, Eberhard
中科院分区：
文献类型：
--
作者：
Ploen, Daniela;Hafirassou, Mohamed Lamine;Hildt, Eberhard
背景 82 目的：丙型肝炎病毒 (HCV) 复制/形态发生发生在膜网上。病毒基因组复制发生在 ER 细胞质面上的复制子复合体中，而 HCV 组装则位于脂滴 (LD) 表面。这就提出了一个问题，即从头合成的病毒基因组从复制子复合体靶向LD和参与该过程的细胞蛋白，例如LD相关蛋白TIP47，也称为细胞质分选因子。 方法：使用感染性HCV JHF1培养系统在HuH7.5细胞中研究病毒复制。通过二维凝胶电泳和质谱法进行蛋白质组分析。通过siRNA或慢病毒转导来调节靶基因的表达。进行共聚焦显微镜分析亚细胞区室。通过免疫共沉淀、亲和层析和酵母双杂交筛选来研究蛋白质/蛋白质相互作用。结果：基于蛋白质组的分析表明，与对照细胞相比，HCV 复制细胞含有较少的 TIP47。然而，表达分析表明 HCV 复制细胞中 TIP47 表达增加。 TIP47 与负载 RNA 的 NS5A 结合。结合域的图谱显示 NS5A 与 TIP47 的 N 端 PAT 域结合。 TIP47 的过度表达会增加释放的病毒数量，而 TIP47 的沉默会减少释放的感染性颗粒的数量。完全敲除TIP47表达可消除病毒复制。结论：TIP47在HCV生命周期中发挥重要作用。 (C) 2013 年欧洲肝脏研究协会。由 Elsevier B.V. 出版。保留所有权利。
Background 82 Aims: Hepatitis C virus (HCV) replication/morphogenesis takes place at the membranous web. Viral genome replication occurs in replicon complexes on the cytoplasmic face of the ER whereas HCV assembly is located on the surface of lipid droplets (LDs). This raises the question about targeting of de novo synthesized viral genomes from the replicon complex to LDs and cellular proteins involved in this process such as the LD-associated protein TIP47, also known as cytoplasmic sorting factor.Methods: Viral replication was studied in HuH7.5 cells using the infectious HCV JHF1 culture system. Proteome analysis was performed by 2D gel electrophoresis and mass spectrometry. Expression of target genes was modulated by siRNA or lentiviral transduction. Confocal microscopy was performed for analysis of subcellular compartments. Protein/protein interactions were studied by co-immunoprecipitations, affinity chromatography, and yeast two-hybrid screens.Results: Proteome based analysis revealed that HCV replicating cells contain less TIP47 compared to control cells. However, expression analyses demonstrated an increased TIP47 expression in HCV replicating cells. TIP47 binds to RNA-loaded NS5A. Mapping of the binding domain revealed that NS5A binds to the N-terminal PAT domain of TIP47. Overexpression of TIP47 increases the amount of released viruses, while silencing of TIP47 decreases the amount of released infectious particles. Complete knockdown of TIP47 expression abolishes virus replication.Conclusions: TIP47 plays an essential role in the HCV life cycle. (C) 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.