Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
Department of Immunoparasitology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
J Virol. 2019 Mar 5;93(6). doi: 10.1128/JVI.01708-18. Print 2019 Mar 15.
Hepatitis C virus (HCV) utilizes cellular factors for efficient propagation. Ubiquitin is covalently conjugated to the substrate to alter its stability or to modulate signal transduction. In this study, we examined the importance of ubiquitination for HCV propagation. We found that inhibition of deubiquitinating enzymes (DUBs) or overexpression of nonspecific DUBs impaired HCV replication, suggesting that ubiquitination regulates HCV replication. To identify specific DUBs involved in HCV propagation, we set up RNA interference (RNAi) screening against DUBs and successfully identified ubiquitin-specific protease 15 (USP15) as a novel host factor for HCV propagation. Our studies showed that USP15 is involved in translation of HCV RNA and production of infectious HCV particles. In addition, deficiency of USP15 in human hepatic cell lines (Huh7 and Hep3B/miR-122 cells) but not in a nonhepatic cell line (293T cells) impaired HCV propagation, suggesting that USP15 participates in HCV propagation through the regulation of hepatocyte-specific functions. Moreover, we showed that loss of USP15 had no effect on innate immune responses and We also found that USP15-deficient Huh7 cells showed reductions in the amounts of lipid droplets (LDs), and the addition of palmitic acids restored the production of infectious HCV particles. Taken together, these data suggest that USP15 participates in HCV propagation by regulating the translation of HCV RNA and the formation of LDs. Although ubiquitination has been shown to play important roles in the HCV life cycle, the roles of deubiquitinating enzymes (DUBs), which cleave ubiquitin chains from their substrates, in HCV propagation have not been investigated. Here, we identified USP15 as a DUB regulating HCV propagation. USP15 showed no interaction with viral proteins and no participation in innate immune responses. Deficiency of USP15 in Huh7 cells resulted in suppression of the translation of HCV RNA and reduction in the amounts of lipid droplets, and the addition of fatty acids partially restored the production of infectious HCV particles. These data suggest that USP15 participates in HCV propagation in hepatic cells through the regulation of viral RNA translation and lipid metabolism.
丙型肝炎病毒（HCV）利用细胞因子进行高效繁殖。泛素被共价连接到底物上，以改变其稳定性或调节信号转导。在这项研究中，我们研究了泛素化在 HCV 繁殖中的重要性。我们发现，去泛素化酶（DUBs）的抑制或非特异性 DUBs 的过表达会损害 HCV 的复制，这表明泛素化调节 HCV 的复制。为了鉴定参与 HCV 繁殖的特定 DUBs，我们针对 DUBs 进行了 RNA 干扰（RNAi）筛选，并成功鉴定出泛素特异性蛋白酶 15（USP15）是 HCV 繁殖的一种新的宿主因子。我们的研究表明，USP15 参与 HCV RNA 的翻译和感染性 HCV 颗粒的产生。此外，USP15 缺陷的人肝细胞系（Huh7 和 Hep3B/miR-122 细胞）而不是非肝细胞系（293T 细胞）中 HCV 的繁殖受损，这表明 USP15 通过调节肝细胞特异性功能参与 HCV 的繁殖。此外，我们表明 USP15 的缺失对先天免疫反应没有影响，我们还发现 USP15 缺陷的 Huh7 细胞中脂滴（LDs）的量减少，添加棕榈酸可恢复感染性 HCV 颗粒的产生。总之，这些数据表明，USP15 通过调节 HCV RNA 的翻译和 LDs 的形成参与 HCV 的繁殖。尽管泛素化已被证明在 HCV 生命周期中发挥重要作用，但尚未研究去泛素化酶（DUBs）在 HCV 繁殖中的作用，这些酶从其底物上切割泛素链。在这里，我们鉴定了 USP15 作为调节 HCV 繁殖的 DUB。USP15 与病毒蛋白没有相互作用，也不参与先天免疫反应。Huh7 细胞中 USP15 的缺失导致 HCV RNA 翻译受到抑制，脂滴数量减少，添加脂肪酸可部分恢复感染性 HCV 颗粒的产生。这些数据表明，USP15 通过调节病毒 RNA 翻译和脂质代谢参与肝细胞中的 HCV 繁殖。
