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. 2012;7(10):e46631.
doi: 10.1371/journal.pone.0046631. Epub 2012 Oct 22.

Iron regulator hepcidin exhibits antiviral activity against hepatitis C virus

Hongyan Liu  1 , Thu Le Trinh, Huijia Dong, Robertson Keith, David Nelson, Chen Liu
Affiliations

Iron regulator hepcidin exhibits antiviral activity against hepatitis C virus

Hongyan Liu et al. PLoS One. 2012.

Abstract

Hepatitis C viral infection affects 170 million people worldwide. It causes serious chronic liver diseases. HCV infection has been implicated in iron accumulation in the liver and iron overload has been shown to be a potential cofactor for HCV associated hepatocellular carcinoma progression. The underlying mechanisms are not understood. Human hepcidin, a 25 amino acid peptide mainly produced by hepatocytes, is a key regulator of iron metabolism. Alteration of hepcidin expression levels has been reported in the setting of chronic HCV infection and hepatocellular carcinoma. In this study, we aim to examine the interactions between HCV infection and hepcidin expression in liver cells. We found that hepcidin expression was suppressed in HCV infected cells. The suppressive effect appears to be regulated by histone acetylation but not DNA methylation. Moreover, we found that hepcidin had a direct antiviral activity against HCV replication in cell culture. The antiviral effect is associated with STAT3 activation. In conclusion, hepcidin can induce intracellular antiviral state while HCV has a strategy to suppress hepcidin expression. This may be a novel mechanism by which HCV circumvents hepatic innate antiviral defense.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Hepcidin expression is decreased in HCV positive cells or HCV replicon cells.
RT-PCR analysis (A) and qRT-PCR analysis (B) demonstrated hepcidin mRNA expression in control cells (Huh7.5, primary hepatocytes[Hep], and Huh7), JFH1 positive cells (Huh7.5 or primary hepatocytes) and HCV replicon cells (FLneo). The data are presented as mean ± SD from three independent experiments.
Figure 2
Figure 2. The regulation of hepcidin expression does not involve DNA methylation.
(A) Huh7.5-JFH1 cells were treated with or without 5-aza-2′-deoxycytidine (Aza), and hepcidin expression was analyzed by qRT-PCR. GAPDH was used as the internal control in the PCR reaction. The data are presented as mean ± SD from three independent experiments; n.s., not significant. (B) and (C) Genomic DNA extracted from Huh7.5, Huh7.5-JFH1 cells (B) or two paired liver tissues (C) was modified with sodium bisulfate, PCR amplified, and subsequently cloned and sequenced. The methylation status at each CpG site in CpG island of hepcidin promoter is shown. Methylated sites are indicated by filled dark circles and unmethylated sites by empty white ones. N: nontumor tissues; T: tumor tissues. (D) Huh7.5-JFH1 cells were treated with or without Trichostatin A (TSA), and hepcidin expression was analyzed by qRT-PCR. The data are presented as mean ± SD from three independent experiments.
Figure 3
Figure 3. Hepcidin peptide inhibits HCV replication.
(A) The synthesized hepcidin peptide with cyclized-disulfide bond between cysteine was shown. (B) and (C) Huh7.5 cells were incubated with JFH1 (B) or HCV-JC1 virus for 16 hours (C), then treated with or hepcidin peptide or control peptide for 3 days, and HCV mRNA levels were analyzed by qRT-PCR. GAPDH was used as the internal control in the PCR reaction. (D) and (E) FLneo cells were treated with or without hepcidin for 3 days and 5 days, and HCV mRNA expression was analyzed by RT-PCR (D) or qRT-PCR (E). The data are presented as mean ± SD from three independent experiments.
Figure 4
Figure 4. Modification of hepcidin expression in Huh7.5 cells affects HCV virus replication.
(A) Western blot analysis of hepcidin expression in Huh7.5-vector, Huh7.5-Hepc and Huh7.5-antiHepc stable cell lines. (B,C) qRT-PCR analysis of HCV mRNA expression in JFH1 infected Huh7.5-vector, Huh7.5-hepc, Huh7.5-antihepc cells (B); and HCV-JC1 infected Huh7.5-vector, Huh7.5-hepc, and Huh7.5-antihepc cells (C). The data are presented as mean ± SD from three independent experiments. (D) Immunostaining for HCV (JC1) NS5A in Huh7.5-vector, Huh7.5-hepc and Huh7.5-antihepc cells on day 7 post infection. The original magnification is 200×. (E) Knockdown of hepcidin mRNA expression in Huh7.5 by shRNA transfection and endogenous hepcidin expression was analysed by qRT-PCR. The effect of hepcidin knockdown on HCV-JFH1 replication was assayed on day 3, day 5 and day 7 post-infection.
Figure 5
Figure 5. The antiviral activity of hepcidin is mediated by STAT3 activation.
(A) Western blot analysis for pSTAT3 and total STAT3 in Huh7.5 control cells and Huh7.5 cells incubated with hepcidin for 0.5 h, 1 h and 4 h. (B) Western blot analysis for STAT3 in Huh7.5-vector, Huh7.5-hepc, Huh7.5-antihepc stable cell lines with or without STAT3 siRNA transfection. Actin was used as the loading control. (C) qRT-PCR analysis for HCV mRNA expression in the cells as described in (B). RNA was prepared from these cells after infected by JC1 virus for 3 days. The data are presented as mean ± SD from three independent experiments.
Figure 6
Figure 6. Hepcidin peptide treatment induces intracellular hepcidin expression.
Huh7.5 cells were incubated with hepcidin for 0.5 h, 1 h and 4 h, followed by total RNA extraction. QRT-PCR was performed to examine hepcidin mRNA expression. The data are presented as mean ± SD from three independent experiments.
Figure 7
Figure 7. Hepcidin induces a classic interferon-induced gene 2′-5′-oligoadenylate synthetase 1 (OAS1) and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) expression.
Huh7.5 cells were infected by HCV-JFH1 virus or JC1 virus, and treated with or without hepcidin peptide for 48 hours. FLneo was also treated with or without hepcidin peptide for 48 hours. OAS1 mRNA levels were analyzed by qRT-PCR in these cells. Huh7.5 cells were infected by HCV-JFH1 virus, and treated with or without hepcidin peptide for 24, 48 and 72 hours. IFIT1 mRNA levels were analyzed by qRT-PCR. GAPDH was used as the internal control in the PCR reaction.
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