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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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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 was arbitrarily defined as 1 in fold increase in HCV, or as 10 in fold increase in miR-122 (A). Cells were infected with JFH-1 for 5 days, and then replated in 6-well plates. After 24 hours, the HCV-infected cells were stimulated with recombinant IFN-λ (100 ng) for 0-48 hours. The amount of HCV RNA or miR-122 was quantified by real time PCR (B, C). Similarly, HCV-infected cells were replated in 6-well plates after 5 days of infection. After 24 hours, cells were transfected with miRIDIAN or negative control miRIDIAN (20 pmol) for 36 h, and stimulated with or without IFN-λ (100 ng each) for 1-3 days. HCV RNA was quantified by real time PCR (D) while HCV NS3 protein was measured by ELISA (E). Data shown are from one experiment and are representative of three experiments with similar results. Statistical significance (p<0.01) is relative to the control group that received no treatment.
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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 was arbitrarily defined as 1 in fold increase in HCV, or as 10 in fold increase in miR-122 (A). Cells were infected with JFH-1 for 5 days, and then replated in 6-well plates. After 24 hours, the HCV-infected cells were stimulated with recombinant IFN-λ (100 ng) for 0-48 hours. The amount of HCV RNA or miR-122 was quantified by real time PCR (B, C). Similarly, HCV-infected cells were replated in 6-well plates after 5 days of infection. After 24 hours, cells were transfected with miRIDIAN or negative control miRIDIAN (20 pmol) for 36 h, and stimulated with or without IFN-λ (100 ng each) for 1-3 days. HCV RNA was quantified by real time PCR (D) while HCV NS3 protein was measured by ELISA (E). Data shown are from one experiment and are representative of three experiments with similar results. Statistical significance (p<0.01) is relative to the control group that received no treatment.

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
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 was arbitrarily defined as 1 in fold increase in HCV, or as 10 in fold increase in miR-122 (A). Cells were infected with JFH-1 for 5 days, and then replated in 6-well plates. After 24 hours, the HCV-infected cells were stimulated with recombinant IFN-λ (100 ng) for 0-48 hours. The amount of HCV RNA or miR-122 was quantified by real time PCR (B, C). Similarly, HCV-infected cells were replated in 6-well plates after 5 days of infection. After 24 hours, cells were transfected with miRIDIAN or negative control miRIDIAN (20 pmol) for 36 h, and stimulated with or without IFN-λ (100 ng each) for 1-3 days. HCV RNA was quantified by real time PCR (D) while HCV NS3 protein was measured by ELISA (E). Data shown are from one experiment and are representative of three experiments with similar results. Statistical significance (p<0.01) is relative to the control group that received no treatment.
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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 was arbitrarily defined as 1 in fold increase in HCV, or as 10 in fold increase in miR-122 (A). Cells were infected with JFH-1 for 5 days, and then replated in 6-well plates. After 24 hours, the HCV-infected cells were stimulated with recombinant IFN-λ (100 ng) for 0-48 hours. The amount of HCV RNA or miR-122 was quantified by real time PCR (B, C). Similarly, HCV-infected cells were replated in 6-well plates after 5 days of infection. After 24 hours, cells were transfected with miRIDIAN or negative control miRIDIAN (20 pmol) for 36 h, and stimulated with or without IFN-λ (100 ng each) for 1-3 days. HCV RNA was quantified by real time PCR (D) while HCV NS3 protein was measured by ELISA (E). Data shown are from one experiment and are representative of three experiments with similar results. Statistical significance (p<0.01) is relative to the control group that received no treatment.
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