Zhu Chuanlong, Xiao Fei, Hong Jian, Wang Kun, Liu Xiao, Cai Dachuan, Fusco Dahlene N, Zhao Lei, Jeong Soung Won, Brisac Cynthia, Chusri Pattranuch, Schaefer Esperance A, Zhao Hong, Peng Lee F, Lin Wenyu, Chung Raymond T
Department of Infectious Disease, Anhui Provincial Hospital, Anhui Medical University, Hefei, Anhui, China Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA Department of Infectious Diseases, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China.
Department of Infectious Disease, Anhui Provincial Hospital, Anhui Medical University, Hefei, Anhui, China Department of Infectious Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
J Virol. 2015 Jul;89(13):6608-18. doi: 10.1128/JVI.00364-15. Epub 2015 Apr 15.
The elongation factor Tu GTP binding domain-containing protein 2 (EFTUD2) was identified as an anti-hepatitis C virus (HCV) host factor in our recent genome-wide small interfering RNA (siRNA) screen. In this study, we sought to further determine EFTUD2's role in HCV infection and investigate the interaction between EFTUD2 and other regulators involved in HCV innate immune (RIG-I, MDA5, TBK1, and IRF3) and JAK-STAT1 pathways. We found that HCV infection decreased the expression of EFTUD2 and the viral RNA sensors RIG-I and MDA5 in HCV-infected Huh7 and Huh7.5.1 cells and in liver tissue from in HCV-infected patients, suggesting that HCV infection downregulated EFTUD2 expression to circumvent the innate immune response. EFTUD2 inhibited HCV infection by inducing expression of the interferon (IFN)-stimulated genes (ISGs) in Huh7 cells. However, its impact on HCV infection was absent in both RIG-I knockdown Huh7 cells and RIG-I-defective Huh7.5.1 cells, indicating that the antiviral effect of EFTUD2 is dependent on RIG-I. Furthermore, EFTUD2 upregulated the expression of the RIG-I-like receptors (RLRs) RIG-I and MDA5 to enhance the innate immune response by gene splicing. Functional experiments revealed that EFTUD2-induced expression of ISGs was mediated through interaction of the EFTUD2 downstream regulators RIG-I, MDA5, TBK1, and IRF3. Interestingly, the EFTUD2-induced antiviral effect was independent of the classical IFN-induced JAK-STAT pathway. Our data demonstrate that EFTUD2 restricts HCV infection mainly through an RIG-I/MDA5-mediated, JAK-STAT-independent pathway, thereby revealing the participation of EFTUD2 as a novel innate immune regulator and suggesting a potentially targetable antiviral pathway.
Innate immunity is the first line defense against HCV and determines the outcome of HCV infection. Based on a recent high-throughput whole-genome siRNA library screen revealing a network of host factors mediating antiviral effects against HCV, we identified EFTUD2 as a novel innate immune regulator against HCV in the infectious HCV cell culture model and confirmed that its expression in HCV-infected liver tissue is inversely related to HCV infection. Furthermore, we determined that EFTUD2 exerts its antiviral activity mainly through governing its downstream regulators RIG-I and MDA5 by gene splicing to activate IRF3 and induce classical ISG expression independent of the JAT-STAT signaling pathway. This study broadens our understanding of the HCV innate immune response and provides a possible new antiviral strategy targeting this novel regulator of the innate response.
在我们最近的全基因组小干扰RNA（siRNA）筛选中，含延伸因子Tu GTP结合结构域蛋白2（EFTUD2）被鉴定为一种抗丙型肝炎病毒（HCV）宿主因子。在本研究中，我们试图进一步确定EFTUD2在HCV感染中的作用，并研究EFTUD2与参与HCV固有免疫（RIG-I、MDA5、TBK1和IRF3）及JAK-STAT1途径的其他调节因子之间的相互作用。我们发现，HCV感染降低了HCV感染的Huh7和Huh7.5.1细胞以及HCV感染患者肝组织中EFTUD2以及病毒RNA传感器RIG-I和MDA5的表达，这表明HCV感染下调EFTUD2表达以规避固有免疫反应。EFTUD2通过诱导Huh7细胞中干扰素（IFN）刺激基因（ISG）的表达来抑制HCV感染。然而，在RIG-I敲低的Huh7细胞和RIG-I缺陷的Huh7.5.1细胞中，其对HCV感染均无影响，这表明EFTUD2的抗病毒作用依赖于RIG-I。此外，EFTUD2通过基因剪接上调RIG-I样受体（RLR）RIG-I和MDA5的表达以增强固有免疫反应。功能实验表明，EFTUD2诱导的ISG表达是通过EFTUD2下游调节因子RIG-I、MDA5、TBK1和IRF3的相互作用介导的。有趣的是，EFTUD2诱导的抗病毒作用独立于经典的IFN诱导的JAK-STAT途径。我们的数据表明，EFTUD2主要通过RIG-I/MDA5介导的、不依赖JAK-STAT的途径限制HCV感染，从而揭示了EFTUD2作为一种新型固有免疫调节因子的参与情况，并提示了一条潜在的可靶向抗病毒途径。
固有免疫是对抗HCV的第一道防线，并决定HCV感染的结果。基于最近一项高通量全基因组siRNA文库筛选揭示了介导抗HCV抗病毒作用的宿主因子网络，我们在感染性HCV细胞培养模型中鉴定EFTUD2为一种新型抗HCV固有免疫调节因子，并证实其在HCV感染肝组织中的表达与HCV感染呈负相关。此外，我们确定EFTUD2主要通过基因剪接调控其下游调节因子RIG-I和MDA5来激活IRF3并诱导经典ISG表达，且不依赖JAT-STAT信号通路，从而发挥其抗病毒活性。本研究拓宽了我们对HCV固有免疫反应的理解，并提供了一种针对这种新型固有免疫调节因子的可能新抗病毒策略。
