Transcriptional regulation of IFN-λ genes in Hepatitis C virus-infected hepatocytes via IRF-3·IRF-7·NF-κB complex

Abstract

Hepatitis C virus (HCV) infection in hepatocytes stimulates innate antiviral responses including the production of type III interferons (IFN-λ), including IL-28A, IL-28B, and IL-29 (Figure ​(Figure1).1). However, the molecular mechanism(s) regulating the expression of IFN-λ genes in HCV-infected hepatocytes remains undefined. In this study, we examined regulatory elements involved in the induction of IFN-λ genes following HCV infection in hepatocytes and further determined the binding of specific transcription factor(s) to promoter regions of IFN-λ genes. Our studies reveal that the regulatory portion for IL-28A, IL-28B, andIL-29 genes is localized to a 1-kb region in their respective promoters (Figure ​(Figure2,2, ​,4).4). Notably, interferon regulatory factor (IRF)-3 and -7 are the key transcriptional factors for the induction of IL-28A and IL-28Bgenes (Figure ​(Figure5,5, ​,6),6), whereas NF-κB is an additional requirement for the induction of the IL-29 gene (Figure ​(Figure3).3). Ligation of Toll-like receptors (TLR) 3, 7, 8, and 9, which also activate IRFs and NF-κB, resulted in more robust production of IFN-λ than that observed with HCV infection, verifying the importance of TLR pathways in IFN-λ production (Figure ​(Figure8).8). Furthermore, the addition of IFN-λ to HCV-infected hepatocytes decreased viral replication and produced a concurrent reduction in microRNA-122 (miR-122). The decrease in viral replication was enhanced by the co-administration of IFN-λ and miR-122 inhibitor (miRIDIAN) (Figure ​(Figure7),7), suggesting that such combinatorial therapies may be beneficial for the treatment of chronic HCV infection. Figure 1 Hepatic induction of IFN-λ genes during HCV infection. A, B. Total RNA was extracted from liver tissues of healthy and chronic HCV patients for IL-28A/B (A) and IL-29 (B) mRNA quantification by real-time PCR. C-F. Human primary hepatocytes were ... Figure 2 Transcriptional activity at the IL-28A, IL-28B, and IL-29 promoter by HCV infection. PH5CH8 cells were transfected with a mixture of the luciferase constructs containing various 5' deletions of the human IL-28A (A), IL-28B (B), or IL-29 (C) promoters ... Figure 3 IRF-7, and NF-κB differentially determine expression of IL-28A/B and IL-29. Nucleotide sequence from 1.0 kb upstream of the transcription start site of human IL-28A, IL-28B, and IL-29. The positions of the IRF, NF-κB, AP-1, and GATA-1 ... Figure 4 Overexpression of IRFs and NF-κB confirms their role in expression of IFN-λ genes. PH5CH8 cells were transiently transfected with plasmids encoding different promoter regions of human IL-28A (pGL3-IL-28A 1kb) (A-C), IL-28B (pGL3-IL-28B ... Figure 5 IRFs are core transcription factors of IFN-λ genes. IL-28B (B), or IL-29 (C) in combination with siRNA specific for IRF-3, IRF-7, RELA(NF-κB p65), or control. At 36 h post-transfection, cells were stimulated with poly(I:C) for 6 hours ... Figure 6 IRFs and NF-κB directly bind the promoters of IFN-λ genes. PH5CH8 cells were stimulated with poly(I:C) (A and C) or infected with JFH-1 (B and D) for the indicated lengths of time. Chromatin was immunoprecipitated with anti-IRF-3, anti-IRF-7, ... Figure 7 miR-122-dependent replication of HCV is suppressed by IFN-λ stimulation. Huh 7.5.1 cells infected with JFH-1 for the indicated number of days and HCV RNA or miR-122 levels were quantified by real time PCR. The mean value of day 1 post-infection ... Figure 8 TLR3, 7, 8, and 9 induce human IFN-λ gene expression in human primary hepatocytes. Human primary hepatocytes were incubated with JFH-1, Pam3CSK4 (1 μg/ml), HKLM (108 cells/ml), Poly (I-C) (5 μg/ml), LPS (10 μg/ml), Flagellin ...