Li Kui, Chen Zihong, Kato Nobuyuki, Gale Michael, Lemon Stanley M
Department of Microbiology and Immunology, Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, Texas 77555-1019, USA.
J Biol Chem. 2005 Apr 29;280(17):16739-47. doi: 10.1074/jbc.M414139200. Epub 2005 Feb 28.
Innate cellular antiviral defenses are likely to influence the outcome of infections by many human viruses, including hepatitis B and C viruses, agents that frequently establish persistent infection leading to chronic hepatitis, cirrhosis, and liver cancer. However, little is known of the pathways by which hepatocytes, the cell type within which these hepatitis agents replicate, sense infection, and initiate protective responses. We show that cultured hepatoma cells, including Huh7 cells, do not activate the interferon (IFN)-beta promoter in response to extracellular poly(I-C). In contrast, the addition of poly(I-C) to culture media activates the IFN-beta promoter and results in robust expression of IFN-stimulated genes (ISG) in PH5CH8 cells, which are derived from non-neoplastic hepatocytes transformed with large T antigen. Small interfering RNA knockdown of TLR3 or its adaptor, Toll-interleukin-1 receptor domain-containing adaptor inducing IFN-beta (TRIF), blocked extracellular poly(I-C) signaling in PH5CH8 cells, whereas poly(I-C) responsiveness could be conferred on Huh7 hepatoma cells by ectopic expression of Toll-like receptor 3 (TLR3). In contrast to poly(I-C), both cell types signal the presence of Sendai virus infection through a TLR3-independent intracellular pathway requiring expression of retinoic acid-inducible gene I (RIG-I), a putative cellular RNA helicase. Silencing of RIG-I expression impaired only the response to Sendai virus and not extracellular poly(I-C). We conclude that hepatocytes contain two distinct antiviral signaling pathways leading to expression of type I IFNs, one dependent upon TLR3 and the other dependent on RIG-I, with little cross-talk between these pathways.
先天性细胞抗病毒防御可能会影响许多人类病毒感染的结果，包括乙型和丙型肝炎病毒，这些病原体经常导致持续性感染，进而引发慢性肝炎、肝硬化和肝癌。然而，对于这些肝炎病原体在其中复制的细胞类型——肝细胞感知感染并启动保护性反应的途径，我们却知之甚少。我们发现，包括Huh7细胞在内的培养肝癌细胞不会因细胞外聚肌苷酸胞苷酸（poly(I-C)）而激活干扰素（IFN）-β启动子。相反，向培养基中添加poly(I-C)会激活PH5CH8细胞中的IFN-β启动子，并导致干扰素刺激基因（ISG）的强劲表达，PH5CH8细胞源自用大T抗原转化的非肿瘤性肝细胞。用小干扰RNA敲低Toll样受体3（TLR3）或其接头含Toll白细胞介素-1受体结构域的接头诱导干扰素-β（TRIF），会阻断PH5CH8细胞中的细胞外poly(I-C)信号传导，而通过异位表达Toll样受体3（TLR3）可使Huh7肝癌细胞获得poly(I-C)反应性。与poly(I-C)不同，两种细胞类型都通过一种不依赖TLR3的细胞内途径来感知仙台病毒感染的存在，该途径需要视黄酸诱导基因I（RIG-I，一种假定的细胞RNA解旋酶）的表达。RIG-I表达的沉默仅损害对仙台病毒的反应，而不影响对细胞外poly(I-C)的反应。我们得出结论，肝细胞包含两条不同的抗病毒信号传导途径，可导致I型干扰素的表达，一条依赖于TLR3，另一条依赖于RIG-I，且这两条途径之间几乎没有相互作用。
