Hepatitis C Virus-Induced ROS/JNK Signaling Pathway Activates the E3 Ubiquitin Ligase Itch to Promote the Release of HCV Particles via Polyubiquitylation of VPS4A
Hepatitis C Virus-Induced ROS/JNK Signaling Pathway Activates the E3 Ubiquitin Ligase Itch to Promote the Release of HCV Particles via Polyubiquitylation of VPS4A
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DOI：
10.1128/jvi.01811-21
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发表时间：
2022-03-23
影响因子：
5.4
通讯作者：
Shoji, Ikuo
中科院分区：
文献类型：
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作者：
Deng, Lin;Liang, Yujiao;Shoji, Ikuo
我们先前报道丙型肝炎病毒(HCV)感染激活了活性氧(ROS)/c-jun氨基末端激酶(JNK)信号通路。然而，ROS/JNK激活在丙型肝炎病毒生命周期中的作用尚不清楚。我们试图确定ROS/JNK信号通路在丙型肝炎病毒生命周期中的新作用。免疫印迹分析显示，丙型肝炎病毒诱导的ROS/JNK激活促进了Hect型E3泛素连接酶Itch的磷酸化，导致Itch的激活。Itch的小干扰RNA(SiRNA)敲除显著降低了细胞外的丙型肝炎病毒感染性滴度、丙型肝炎病毒RNA和丙型肝炎病毒核心蛋白，而不影响细胞内的丙型肝炎病毒感染性滴度、丙型肝炎病毒RNA和丙型肝炎病毒蛋白，提示艾奇参与了丙型肝炎病毒颗粒的释放。丙型肝炎病毒介导的JNK/Itch激活特异性地促进了AAA型ATPase VPS4A的多泛素化，而不是VPS4B，这是形成多囊体所必需的。定点突变发现，VPS4A上的两个赖氨酸残基(K23和K121)对VPS4A的多泛素化起重要作用。VPS4A而不是VPS4B的siRNA敲除显著降低了细胞外丙型肝炎病毒的感染力滴度。免疫共沉淀分析表明，丙型肝炎病毒感染特异性地增强了转运所需的内体分选复合体(ESCRT)-III复合体的亚单位CHMP1B与VPS4A之间的相互作用，但不增强VPS4B与CHMP1B之间的相互作用，而VPS4A K23R/K121R显著降低了与CHMP1B的相互作用。丙型肝炎病毒感染显著增加VPS4A的ATPase活性，但对VPS4A的K23R/K121R和VPS4B无明显影响，提示丙型肝炎病毒介导的VPS4A的多泛素化参与了VPS4A的激活。综上所述，我们认为，丙型肝炎病毒诱导的ROS/JNK/Itch信号通路促进VPS4A多泛素化，导致VPS4A-CHMP1B相互作用增强，促进VPS4A ATPase活性，从而促进丙型肝炎病毒颗粒的释放。ROS/JNK信号通路在肝脏疾病中起重要作用，包括脂肪变性、代谢紊乱和肝细胞癌。我们先前报道，丙型肝炎病毒激活ROS/JNK信号通路，导致肝脏糖异生增强和诱导细胞凋亡。这项研究进一步证明，丙型肝炎病毒诱导的ROS/JNK信号通路激活E3泛素连接酶Itch，通过VPS4A的多泛素化促进丙型肝炎病毒颗粒的释放。我们提供的证据表明，丙型肝炎病毒感染促进ROS/JNK/Itch信号通路和ESCRT/VPS4A机制释放具有感染性的丙型肝炎病毒颗粒。我们的结果可能有助于更好地理解丙型肝炎病毒颗粒释放的机制细节。ROS/JNK信号通路与肝脏疾病有关，包括脂肪变性、代谢紊乱和肝细胞癌。我们先前报道，丙型肝炎病毒激活ROS/JNK信号通路，导致肝脏糖异生增强和诱导细胞凋亡。
We previously reported that hepatitis C virus (HCV) infection activates the reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK) signaling pathway. However, the roles of ROS/JNK activation in the HCV life cycle remain unclear. We sought to identify a novel role of the ROS/JNK signaling pathway in the HCV life cycle. Immunoblot analysis revealed that HCV-induced ROS/JNK activation promoted phosphorylation of Itch, a HECT-type E3 ubiquitin ligase, leading to activation of Itch. The small interfering RNA (siRNA) knockdown of Itch significantly reduced the extracellular HCV infectivity titers, HCV RNA, and HCV core protein without affecting intracellular HCV infectivity titers, HCV RNA, and HCV proteins, suggesting that Itch is involved in the release of HCV particles. HCV-mediated JNK/Itch activation specifically promoted polyubiquitylation of an AAA-type ATPase, VPS4A, but not VPS4B, required to form multivesicular bodies. Site-directed mutagenesis revealed that two lysine residues (K23 and K121) on VPS4A were important for VPS4A polyubiquitylation. The siRNA knockdown of VPS4A, but not VPS4B, significantly reduced extracellular HCV infectivity titers. Coimmunoprecipitation analysis revealed that HCV infection specifically enhanced the interaction between CHMP1B, a subunit of endosomal sorting complexes required for transport (ESCRT)-III complex, and VPS4A, but not VPS4B, whereas VPS4A K23R/K121R greatly reduced the interaction with CHMP1B. HCV infection significantly increased ATPase activity of VPS4A, but not VPS4A K23R/K121R or VPS4B, suggesting that HCV-mediated polyubiquitylation of VPS4A contributes to activation of VPS4A. Taken together, we propose that the HCV-induced ROS/JNK/Itch signaling pathway promotes VPS4A polyubiquitylation, leading to enhanced VPS4A-CHMP1B interaction and promotion of VPS4A ATPase activity, thereby promoting the release of HCV particles. IMPORTANCE The ROS/JNK signaling pathway contributes to liver diseases, including steatosis, metabolic disorders, and hepatocellular carcinoma. We previously reported that HCV activates the ROS/JNK signaling pathway, leading to the enhancement of hepatic gluconeogenesis and apoptosis induction. This study further demonstrates that the HCV-induced ROS/JNK signaling pathway activates the E3 ubiquitin ligase Itch to promote release of HCV particles via polyubiquitylation of VPS4A. We provide evidence suggesting that HCV infection promotes the ROS/JNK/Itch signaling pathway and ESCRT/VPS4A machinery to release infectious HCV particles. Our results may lead to a better understanding of the mechanistic details of HCV particle release.The ROS/JNK signaling pathway contributes to liver diseases, including steatosis, metabolic disorders, and hepatocellular carcinoma. We previously reported that HCV activates the ROS/JNK signaling pathway, leading to the enhancement of hepatic gluconeogenesis and apoptosis induction.