Department of Laboratory Medicine, University of Washington, Harborview Medical Center, Seattle, Washington, USA.
J Virol. 2014 Feb;88(3):1582-90. doi: 10.1128/JVI.02007-13. Epub 2013 Nov 20.
Hepatitis C virus (HCV) infection of hepatocytes leads to transcriptional induction of the chemokine CXCL10, which is considered an interferon (IFN)-stimulated gene. However, we have recently shown that IFNs are not required for CXCL10 induction in hepatocytes during acute HCV infection. Since the CXCL10 promoter contains binding sites for several proinflammatory transcription factors, we investigated the contribution of these factors to CXCL10 transcriptional induction during HCV infection in vitro. Wild-type and mutant CXCL10 promoter-luciferase reporter constructs were used to identify critical sites of transcriptional regulation. The proximal IFN-stimulated response element (ISRE) and NF-κB binding sites positively regulated CXCL10 transcription during HCV infection as well as following exposure to poly(I·C) (a Toll-like receptor 3 [TLR3] stimulus) and 5' poly(U) HCV RNA (a retinoic acid-inducible gene I [RIG-I] stimulus) from two viral genotypes. Conversely, binding sites for AP-1 and CCAAT/enhancer-binding protein β (C/EBP-β) negatively regulated CXCL10 induction in response to TLR3 and RIG-I stimuli, while only C/EBP-β negatively regulated CXCL10 during HCV infection. We also demonstrated that interferon-regulatory factor 3 (IRF3) is transiently recruited to the proximal ISRE during HCV infection and localizes to the nucleus in HCV-infected primary human hepatocytes. Furthermore, IRF3 activated the CXCL10 promoter independently of type I or type III IFN signaling. The data indicate that sensing of HCV infection by RIG-I and TLR3 leads to direct recruitment of NF-κB and IRF3 to the CXCL10 promoter. Our study expands upon current knowledge regarding the mechanisms of CXCL10 induction in hepatocytes and lays the foundation for additional mechanistic studies that further elucidate the combinatorial and synergistic aspects of immune signaling pathways.
丙型肝炎病毒（HCV）感染肝细胞会导致趋化因子 CXCL10 的转录诱导，该趋化因子被认为是干扰素（IFN）刺激基因。然而，我们最近表明，在 HCV 感染期间，IFNs 不是肝细胞中 CXCL10 诱导所必需的。由于 CXCL10 启动子包含多个促炎转录因子的结合位点，我们研究了这些因子在 HCV 感染期间体外 CXCL10 转录诱导的贡献。野生型和突变型 CXCL10 启动子-荧光素酶报告基因构建体用于鉴定转录调控的关键位点。在 HCV 感染以及暴露于聚 I·C（Toll 样受体 3 [TLR3] 刺激物）和 5'聚 U HCV RNA（视黄酸诱导基因 I [RIG-I] 刺激物）后，近端 IFN 刺激反应元件（ISRE）和 NF-κB 结合位点正向调节 CXCL10 转录两种病毒基因型。相反，AP-1 和 CCAAT/增强子结合蛋白 β（C/EBP-β）的结合位点负调节 TLR3 和 RIG-I 刺激物诱导的 CXCL10 诱导，而仅 C/EBP-β在 HCV 感染期间负调节 CXCL10。我们还证明，IRF3 在 HCV 感染期间瞬时募集到近端 ISRE 并在 HCV 感染的原代人肝细胞中定位于核内。此外，IRF3 独立于 I 型或 III 型 IFN 信号转导激活 CXCL10 启动子。该数据表明，RIG-I 和 TLR3 对 HCV 感染的感应导致 NF-κB 和 IRF3 直接募集到 CXCL10 启动子。我们的研究扩展了目前关于肝细胞中 CXCL10 诱导机制的知识，并为进一步阐明免疫信号通路的组合和协同方面的机制研究奠定了基础。
