title: Expression of an IRF-3 fusion protein and mouse estrogen receptor, inhibits hepatitis C viral replication in RIG-I-deficient Huh 7.5 cells.
abstract: Interferon Regulatory Factor-3 (IRF-3) plays a central role in the induction of interferon (IFN) production and succeeding interferon-stimulated genes (ISG) expression en route for restraining hepatitis C virus (HCV) infection. Here, we established a stable Huh7.5-IRF3ER cell line expressing a fusion protein of IRF-3 and mouse estrogen receptor (ER) to examine IFN production and anti-HCV effects of IRF-3 in retinoic acid inducible-gene-I (RIG-I) deficient Huh 7.5 cells. Homodimerization of the IRF-3ER fusion protein was detected by Western blotting after treatment with the estrogen receptor agonist 4-hydrotamoxifen (4-HT) in Huh7.5-IRF3ER cells. Expression of IFN-α, IFN-β, and their inhibitory effects on HCV replication were demonstrated by real-time polymerase chain reaction (PCR). Peak expression of IFN-α and IFN-β was achieved 24-hours post 4-HT treatment, coinciding with the appearance of phosphorylated signal transducer and activator of transcription (STAT) proteins. Additionally, HCV viral replication declined in time-dependent fashion. In previous studies, a novel IFN-mediated pathway regulating expression of 1-8U and heterogeneous nuclear ribonucleoprotein M (hnRNP M) inhibited HCV internal ribosomal entry site (IRES)-dependent translation. When expression of ISGs such as 1-8U and hnRNP M were measured in 4-HT-treated Huh7.5-IRF3ER cells, both genes were positively regulated by activation of the IRF-3ER fusion protein. In conclusion, the anti-HCV effects of IRF-3ER homodimerization inhibited HCV RNA replication as well as HCV IRES-dependent translation in Huh7.5-IRF3ER cells. The results of this study indicate that IRF-3ER homodimerization is a key step to restore IFN expression in Huh7.5-IRF3ER cells and in achieving its anti-HCV effects.
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, University of Florida-Jacksonville, FL 32206, USA.
authors.forename: Luyu
authors.initials: L
authors.lastname: Yao
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Yan
authors.forename: Huijia
authors.initials: H
authors.lastname: Dong
authors.forename: David R
authors.initials: DR
authors.lastname: Nelson
authors.forename: Chen
authors.initials: C
authors.lastname: Liu
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Li
mesh_terms: D000818:Animals; D002460:Cell Line; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000172:deficiency*; D015870:Gene Expression; D016174:Hepacivirus / Q000254:growth & development / Q000276:immunology / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology*; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000096:biosynthesis* / Q000235:genetics; D016898:Interferon-alpha / Q000096:biosynthesis / Q000276:immunology; D016899:Interferon-beta / Q000096:biosynthesis / Q000276:immunology; D051379:Mice; D014176:Protein Biosynthesis; D055503:Protein Multimerization; D060888:Real-Time Polymerase Chain Reaction; D011960:Receptors, Estrogen / Q000096:biosynthesis* / Q000235:genetics; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic; D014779:Virus Replication
chemical_list: D050838:Interferon Regulatory Factor-3; D016898:Interferon-alpha; D011960:Receptors, Estrogen; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; D016899:Interferon-beta; C452820:RIGI protein, human; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases