Tran Si C, Pham Tu M, Nguyen Lam N, Park Eun-Mee, Lim Yun-Sook, Hwang Soon B
National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea.
Korea National Institute of Health, Cheongwon-gun, South Korea.
J Virol. 2016 Jul 27;90(16):7231-7247. doi: 10.1128/JVI.00326-16. Print 2016 Aug 15.
Hepatitis C virus (HCV) infection often causes chronic hepatitis, liver cirrhosis, and ultimately hepatocellular carcinoma. However, the mechanisms underlying HCV-induced liver pathogenesis are still not fully understood. By transcriptome sequencing (RNA-Seq) analysis, we recently identified host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these, tribbles homolog 3 (TRIB3) was selected for further characterization. TRIB3 was initially identified as a binding partner of protein kinase B (also known as Akt). TRIB3 blocks the phosphorylation of Akt and induces apoptosis under endoplasmic reticulum (ER) stress conditions. HCV has been shown to enhance Akt phosphorylation for its own propagation. In the present study, we demonstrated that both mRNA and protein levels of TRIB3 were increased in the context of HCV replication. We further showed that promoter activity of TRIB3 was increased by HCV-induced ER stress. Silencing of TRIB3 resulted in increased RNA and protein levels of HCV, whereas overexpression of TRIB3 decreased HCV replication. By employing an HCV pseudoparticle entry assay, we further showed that TRIB3 was a negative host factor involved in HCV entry. Both in vitro binding and immunoprecipitation assays demonstrated that HCV NS3 specifically interacted with TRIB3. Consequently, the association of TRIB3 and Akt was disrupted by HCV NS3, and thus, TRIB3-Akt signaling was impaired in HCV-infected cells. Moreover, HCV modulated TRIB3 to promote extracellular signal-regulated kinase (ERK) phosphorylation, activator protein 1 (AP-1) activity, and cell migration. Collectively, these data indicate that HCV exploits the TRIB3-Akt signaling pathway to promote persistent viral infection and may contribute to HCV-mediated pathogenesis.
TRIB3 is a pseudokinase protein that acts as an adaptor in signaling pathways for important cellular processes. So far, the functional involvement of TRIB3 in virus-infected cells has not yet been demonstrated. We showed that both mRNA and protein expression levels of TRIB3 were increased in the context of HCV RNA replication. Gene silencing of TRIB3 increased HCV RNA and protein levels, and thus, overexpression of TRIB3 decreased HCV replication. TRIB3 is known to promote apoptosis by negatively regulating the Akt signaling pathway under ER stress conditions. Most importantly, we demonstrated that the TRIB3-Akt signaling pathway was disrupted by NS3 in HCV-infected cells. These data provide evidence that HCV modulates the TRIB3-Akt signaling pathway to establish persistent viral infection.
丙型肝炎病毒（HCV）感染常导致慢性肝炎、肝硬化，最终发展为肝细胞癌。然而，HCV诱导肝脏发病机制仍未完全明确。通过转录组测序（RNA-Seq）分析，我们最近鉴定出在细胞培养增殖的HCV（HCVcc）感染细胞中显著差异表达的宿主基因。其中，选择TRIB3（TRIBbles同源物3）进行进一步研究。TRIB3最初被鉴定为蛋白激酶B（也称为Akt）的结合伴侣。TRIB3可阻断Akt的磷酸化，并在内质网（ER）应激条件下诱导细胞凋亡。已有研究表明，HCV为自身增殖增强Akt磷酸化。在本研究中，我们证实HCV复制时TRIB3的mRNA和蛋白水平均升高。我们进一步表明，HCV诱导的ER应激增加了TRIB3的启动子活性。沉默TRIB3导致HCV的RNA和蛋白水平升高，而TRIB3过表达则降低HCV复制。通过使用HCV假病毒颗粒进入试验，我们进一步表明TRIB3是参与HCV进入的负性宿主因子。体外结合试验和免疫沉淀试验均表明，HCV NS3特异性与TRIB3相互作用。因此，HCV NS3破坏了TRIB3与Akt的结合，从而在HCV感染细胞中损害了TRIB3-Akt信号传导。此外，HCV调节TRIB3以促进细胞外信号调节激酶（ERK）磷酸化、激活蛋白1（AP-1）活性和细胞迁移。总体而言，这些数据表明HCV利用TRIB3-Akt信号通路促进持续性病毒感染，并可能导致HCV介导的发病机制。
TRIB3是一种伪激酶蛋白，在重要细胞过程的信号通路中作为衔接子发挥作用。到目前为止，TRIB3在病毒感染细胞中的功能尚未得到证实。我们表明，在HCV RNA复制的情况下，TRIB3的mRNA和蛋白表达水平均升高。TRIB3基因沉默增加了HCV RNA和蛋白水平，因此，TRIB3过表达降低了HCV复制。已知TRIB3在ER应激条件下通过负调节Akt信号通路促进细胞凋亡。最重要的是，我们证明在HCV感染细胞中，NS3破坏了TRIB3-Akt信号通路。这些数据提供了证据，表明HCV调节TRIB3-Akt信号通路以建立持续性病毒感染。
