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

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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 binding sites are indicated. (B-D) PH5CH8 cells were transiently transfected with control plasmid pGL3-basic, and plasmids encoding luciferase constructs containing human IL-28A (pGL3-IL-28A 1kb) (B), IL-28B (pGL3-IL-28B 1kb) (C), or IL-29 (pGL3-IL-29 1kb) (D) promoter regions. Various mutation constructs of the above promoter regions were also transfected into PH5CH8 cells, using a Renilla luciferase vector as a control. The cells were cultured for 24 hours and then stimulated with poly(I:C) for 6 h or infected with JFH-1 for 12 h. Luciferase activity in whole cell lysates was normalized to Renilla luciferase activity. Data are the mean ± SD of triplicate data points from five independent experiments.
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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 binding sites are indicated. (B-D) PH5CH8 cells were transiently transfected with control plasmid pGL3-basic, and plasmids encoding luciferase constructs containing human IL-28A (pGL3-IL-28A 1kb) (B), IL-28B (pGL3-IL-28B 1kb) (C), or IL-29 (pGL3-IL-29 1kb) (D) promoter regions. Various mutation constructs of the above promoter regions were also transfected into PH5CH8 cells, using a Renilla luciferase vector as a control. The cells were cultured for 24 hours and then stimulated with poly(I:C) for 6 h or infected with JFH-1 for 12 h. Luciferase activity in whole cell lysates was normalized to Renilla luciferase activity. Data are the mean ± SD of triplicate data points from five independent experiments.

Mentions: 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 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 2, 4). Notably, interferon regulatory factor (IRF)-3 and -7 are the key transcriptional factors for the induction of IL-28A and IL-28Bgenes (Figure 5, 6), whereas NF-κB is an additional requirement for the induction of the IL-29 gene (Figure 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 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 7), suggesting that such combinatorial therapies may be beneficial for the treatment of chronic HCV infection.


Transcriptional regulation of IFN- λ genes in Hepatitis C virus-infected hepatocytes via IRF-3 · IRF-7 · NF- κ B complex
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 binding sites are indicated. (B-D) PH5CH8 cells were transiently transfected with control plasmid pGL3-basic, and plasmids encoding luciferase constructs containing human IL-28A (pGL3-IL-28A 1kb) (B), IL-28B (pGL3-IL-28B 1kb) (C), or IL-29 (pGL3-IL-29 1kb) (D) promoter regions. Various mutation constructs of the above promoter regions were also transfected into PH5CH8 cells, using a Renilla luciferase vector as a control. The cells were cultured for 24 hours and then stimulated with poly(I:C) for 6 h or infected with JFH-1 for 12 h. Luciferase activity in whole cell lysates was normalized to Renilla luciferase activity. Data are the mean ± SD of triplicate data points from five independent experiments.
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Related In: Results  -  Collection

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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 binding sites are indicated. (B-D) PH5CH8 cells were transiently transfected with control plasmid pGL3-basic, and plasmids encoding luciferase constructs containing human IL-28A (pGL3-IL-28A 1kb) (B), IL-28B (pGL3-IL-28B 1kb) (C), or IL-29 (pGL3-IL-29 1kb) (D) promoter regions. Various mutation constructs of the above promoter regions were also transfected into PH5CH8 cells, using a Renilla luciferase vector as a control. The cells were cultured for 24 hours and then stimulated with poly(I:C) for 6 h or infected with JFH-1 for 12 h. Luciferase activity in whole cell lysates was normalized to Renilla luciferase activity. Data are the mean ± SD of triplicate data points from five independent experiments.
Mentions: 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 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 2, 4). Notably, interferon regulatory factor (IRF)-3 and -7 are the key transcriptional factors for the induction of IL-28A and IL-28Bgenes (Figure 5, 6), whereas NF-κB is an additional requirement for the induction of the IL-29 gene (Figure 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 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 7), suggesting that such combinatorial therapies may be beneficial for the treatment of chronic HCV infection.

View Article: PubMed Central - HTML

No MeSH data available.


Related in: MedlinePlus