Elevated levels of iron ion and hepcidin expression in HCV-infected cells

To determine how HCV infection affects iron levels in host cells, the Nitroso-PSAP method (Fig 1A) and FeRhoNox-1 fluorescence intensity (Fig 1B) were used to quantitatively determine the concentrations of free ferrous and ferric iron in HCV-infected cells. An increase in intracellular iron concentration was observed after virus infection in a titer-dependent manner. Increased iron concentrations due to HCV infection were also observed by histological detection specific to ferrous iron with a turn-on fluorescent probe (Fig 1B). When the gene expression of iron regulation-related factors such as hepcidin, ferroportin 1 (FPN1), divalent metal transporter 1 (DMT1), transferrin receptor 1 (TfR1) and TfR2 and ferritin were compared in HCV-infected and -uninfected cells, the increase in expression of the hepcidin gene due to infection was most pronounced, with expression increasing up to 5-fold or more in infected cells compared to uninfected cells (Fig 1C). The expression level of ferritin was unchanged or increased only slightly by HCV infection (S1 Fig). This appears to be consistent with around 1.4-fold change in intracellular iron content in HCV infection (Fig 1A and 1B). The induction of hepcidin gene expression by HCV infection was observed at 24 h post-infection (hpi), with a marked increase in its expression proportional to the amount of viral infection at 48 hpi (Fig 1D). As a control experiment, we inoculated a concentrate of the culture supernatant of uninfected cells and confirmed that it did not affect hepcidin expression as well as the mock control (S2 Fig). Analysis of HCV-infected long-term culture systems in which Huh7.5.1 cells can continue to be cultured without passaging for several weeks in the presence of DMSO [19,20] also showed increased expression of the hepcidin gene and intracellular iron accumulation in cells at 7, 14, and 21 days of HCV infection (Fig 1E).

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Fig 1. Induction of hepcidin gene expression and intracellular iron accumulation by HCV infection.

Iron concentrations in HCV-infected Huh7.5.1 cells 3 days post-infection (dpi) at MOIs of 0, 0.5, and 3 were evaluated through (A) measurement of Fe²⁺ and Fe³⁺ in cell lysates using the Nitroso-PSAP method and (B) measurement of relative intensity corresponding to Fe²⁺ using the fluorescent probe FeRhoNox-1. (B) For each group, cytoplasmic luminescence intensities of cells stained with the fluorescence probe were measured in Huh7.5.1 cells and the relative intensities were graphed with the average intensity of the uninfected-cell group as 1. Representative images of the cells used to measure luminescence intensities are shown at the bottom of the graph. (C) mRNA expressions of hepcidin, FPN1, DMT1, TfR1, TfR2 and ferritin in infected cells 3 dpi (MOI = 0.5, HCV+) and cells without infection (HCV-) were analyzed using qRT-PCR. (D) Time-course changes in hepcidin gene expression induced by HCV infection at MOIs of 1 and 3 were determined. At 12, 24, and 48 hpi, hepcidin mRNA was quantified as above. (E) In long-term cultures of infected cells, hepcidin mRNA was measured at 7, 14, and 21 dpi at MOI of 0.5. Values obtained from mock-infections at each point were set as 1. Intracellular iron concentrations were determined by the Nitroso-PSAP. (A)-(E) Results represent the means with SD from three independent measurements. Student’s t test; *P<0.05, **P<0.01, ***P <0.001.

https://doi.org/10.1371/journal.ppat.1011591.g001

Critical role of CREBH in the transcriptional upregulation of BMP6 and hepcidin caused by HCV infection

It has been reported that transcription factors such as SMAD1 and CREBH bind to the hepcidin promoter sequence, thereby positively regulating its gene expression [10–12]. We analyzed the effect of HCV infection on the recruitment of transcription factors to the hepcidin promoter in cells by chromatin immunoprecipitation (ChIP). The amplified DNA fragments containing the CREBH binding site region in nt -139/-132 (TGACACAA) and BMP-RE region in nt -84/-79 (GGCGCC) were detected after immunoprecipitation with anti-CREBH and anti-SMAD1 antibodies, respectively. These DNA levels were clearly higher in HCV-infected cells than in uninfected cells (Fig 2A).

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Fig 2. Role of CREBH in hepcidin and BMP6 expression induced by HCV infection or protein expression.

(A) Nuclear fractions from infected cells 3 dpi (MOI = 0.5) and cells without infection were used for ChIP assays. Protein-DNA complexes were immunoprecipitated with antibodies against either SMAD1 or CREBH or with rabbit IgG, analyzed by qPCR using three primer sets: Hepcidin pr ChIP set #1-#3. The sites potentially recognized by CREBH or BMP6 are indicated. (B) BMP6 mRNA in cells with and without HCV infection was analyzed using qRT-PCR. (C) BMP6 promoter reporter (BMP6pr) or basal reporter (EV; empty vector) was transfected into cells with HCV infection at 1 dpi and without infection. Firefly luciferase activities normalized by Renilla luciferase in cells at 2 days post-transfection (dpt) were determined. (D) ChIP assays and immunoprecipitations were performed as described in (A) with anti-CREBH antibody against or rabbit IgG. Precipitates were analyzed for qPCR using two primer sets: BMP6 pr ChIP set #1 and #2. The CREBH-binding sites are indicated. (E) CREBH-KO and parental Huh7.5.1 cells were cultured with or without HCV infection for 3 days, after which the mRNA of hepcidin (left) and BMP6 (middle) were quantified and the iron concentration (right) was measured by the Nitroso-PSAP. (F) Cells were transfected with a plasmid expressing Core-NS2 or NS3-5B or an EV. At 2 dpt, mRNAs of hepcidin, BMP6, and GAPDH were quantified. (G) Eight-week-old male WT and Creb3l3^−/− mice were intravenously injected with AdV expressing Cre recombinase (AdEFNCre) together with AdV expressing either GFP (AdEFLNLGFP) or Core-NS2 (AdEFLNLHCVCore/NS2). At 5 dpi, total RNAs from liver tissues were extracted, followed by determining hepcidin and BMP6 mRNAs. Expression of HCV RNA was detected by RT-PCR. (B, E-G) Results represent the means with SD from three independent measurements. Student’s t test; *P<0.05, **P<0.01, ***P <0.001.

https://doi.org/10.1371/journal.ppat.1011591.g002

Bone morphogenetic proteins (BMPs) are members of the TGFβ superfamily, and their binding to BMP type I receptor triggers activation of the downstream Smad1/5/8 signaling cascade, leading to transcription of target genes [21]. The BMP6-SMAD signaling pathway is a central regulator of hepcidin expression. We observed marked increases in hepcidin mRNA expression 6 h after the addition of recombinant BMP6 to Huh7.5.1 cells (S3A Fig) and in BMP6 mRNA expression in HCV-infected cells (Fig 2B). Enhancement of BMP6 promoter activity by HCV infection was also found using a luciferase reporter assay (Fig 2C). Introduction of a BMP6-expressing plasmid into the cells increased hepcidin mRNA expression (S3B Fig), while introduction of BMP6 siRNA decreased hepcidin mRNA expression (S3C Fig). These results indicate that BMP6 expressed in hepatocyte-derived cells is potentially involved in the regulation of hepcidin expression.

Transcription factor binding site prediction revealed that there are two nucleotide sequences mimicking the CRE consensus site on the BMP6 promoter at -1,123/-1,115 (CCACGTGT) and -667/-660 (CCACGTTT). ChIP assay was used to analyze whether CREBH binds to these consensus-like sites, and if so, whether HCV infection affects CREBH-BMP6 promoter binding. Quantitative PCR using CREBH antibody precipitates as templates showed that among the two CRE-like sequences, the -667/-660 site within the BMP6 promoter is likely to interact with CREBH, and that this CREBH-BMP6 promoter interaction is enhanced by HCV infection (Fig 2D).

The involvement of CREBH in the increased expression of BMP6 and hepcidin by HCV infection was further analyzed using CREBH-knockout (CREBH-KO) cells constructed by the CRISPR-Cas9 system [22]. While CREBH-KO Huh7.5.1 cells showed susceptibility to HCV infection comparable to the parental non-knockout cells, no upregulation of BMP6 or hepcidin mRNA expression by HCV infection was observed (Fig 2E), suggesting that CREBH plays a pivotal role in the regulation of gene expression of BMP6 and hepcidin, especially in their upregulation induced by HCV infection. Coupled with the complete cancellation of the induction of hepcidin expression, the increase in intracellular iron levels associated with HCV infection was also considerably suppressed in CREBH-KO cells. Nevertheless, a moderate increase in iron levels was observed in HCV-infected knockout cells (Fig 2E), suggesting that there may be a hepcidin-independent mechanism for intracellular iron accumulation caused by HCV infection.

In our previous study, we found that CREBH is activated by the expression of the Core-NS2 polyprotein among HCV proteins [23]. As expected, transfection of human hepatoma cells with the Core-NS2 expression plasmid enhanced the expression of BMP6 and hepcidin mRNA, whereas the expression of NS3-NS5B proteins did not show such an increase (Figs 2F and S4). HCV Core-NS2- or GFP-expressing adenovirus (AdV) were injected into the tail veins of C57/BL6J (WT) and CREBH-KO (Creb3l3^−/−) mice to analyze hepcidin and BMP6 expression in liver tissue. We found that HCV core-NS2 expression in WT mice led to increases in hepcidin and BMP6 mRNA expression, but no increase was observed in CREBH-KO mice, indicating that CREBH plays an important role in the induction of HCV-associated hepcidin expression, not only in cultured cells but also in liver tissues (Fig 2G). Since the co-existence of the N-terminal region of the NS3 protein is known to be required for the protease activity of HCV NS2, it is unlikely that CREBH is directly cleaved by NS2 protease activity in the case of Core-NS2 expression. Nevertheless, whether NS2 protease activity may be involved in the proteolytic cleavage of CREBH was tested. We co-expressed the full-length CREBH (CREBH-F) with Core-only, Core-NS2 or Core-NS2 C113S, which contains a mutation in the active center of the NS2 protease, and analyzed CREBH-F cleavage by western blotting (S5 Fig). The result showed that the cleaved fragment of CREBH (CREBH-N) was detected in the expression of Core-NS2 or Core-NS2 C113S more markedly compared to Core-only expression, suggesting that the activation of CREBH is not due to NS2 protease activity. Further analysis was performed to determine whether the induction of CREBH activation by Core-NS2 is due to the activation of general ER stress or unfolded protein response (UPR) which has been shown to activate CREBH [24]. Activation of the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) pathway and inositol requiring enzyme-1 (IRE1) pathway was determined by detection of phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and splicing of X-box binding protein 1 (XBP1) mRNA, respectively (S6A and S6B Fig). Both phosphorylated eIF2α and XBP1 spliced RNA were induced by expression of HCV Core-NS2- or Core-NS2 C113S to the same level as in HCV infection. Their induced levels were clearly greater than that of Core expression alone. Activating transcription factor 6 (ATF6) is known to be a critical determinant of dynamics of transcriptional factor CCAAT-enhancer-binding protein homologous protein (CHOP) during UPR. The expression of Core-NS2- or Core-NS2 C113S resulted in a significant increase in CHOP mRNA expression, whereas the induction of CHOP expression was only limited in the case of Core expression alone (S6C Fig). These results suggest that HCV Core-NS2 is involved in the activation of general ER stress and its activation is independent of NS2 protease activity, and that Core-NS2 expression potentially induces the ER stress more efficiently compared to the case of Core-only expression.

Decrease in serum levels of hepcidin-25 following viral clearance by antiviral therapy in hepatitis C patients

To determine whether upregulation of hepcidin mRNA expression by HCV leads to changes in hepcidin-25 peptide levels, concentrations of hepcidin-25 in the culture supernatants of HCV-infected cells were measured by quantitative liquid chromatography tandem mass chromatography (LC-MS/MS). In Huh7.5.1 cell cultures, the hepcidin-25 level in culture supernatants increased in relation to the viral infection level. In contrast, in CREBH-KO Huh7.5.1 cell cultures in which the viral proteins were expressed as much as in the infected Huh7.5.1 cells, hepcidin-25 concentrations in the supernatants were below the detection limit even at a high infectious titer (MOI = 1) (Fig 3A).

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Fig 3. Hepcidin-25 measurement in culture supernatants with HCV infection and in sera from hepatitis C patients.

(A) Hepcidin-25 concentrations in the supernatants of HCV-infected CREBH-KO cells and parental cells at 3 dpi at MOIs of 0, 0.2, and 1 were measured by the LC-MS/MS. As a positive control (PC), culture supernatants of cells transfected with the hepcidin-expressing plasmid were collected at 2 dpt and used. Expression of HCV Core and GAPDH in cells was assessed. (B) Changes in serum hepcidin-25 concentrations with decreasing viral loads in hepatitis C patients were analyzed. Paired serum samples from 26 hepatitis C patients were collected before (HCV positive) and after DAA therapy (HCV negative), and serum hepcidin-25 concentrations before vs. after viral elimination were compared. Significant differences between two groups before and after therapy were analyzed using Wilcoxon rank sum tests (***P<0.001).

https://doi.org/10.1371/journal.ppat.1011591.g003

Hepcidin has been identified as an acute phase protein induced by inflammatory factors, and it is not always clear how its expression changes in chronic inflammatory conditions. To address this issue and to investigate whether hepcidin-25 levels in human blood are associated with the presence or absence of HCV infection, we quantitated hepcidin-25 in pre-treatment (HCV positive) and post-treatment (HCV negative) serum of hepatitis C patients who had achieved sustained virologic response (SVR) by DAA therapy. Of the 26 paired patient sera measured, 23 patients showed a decrease in serum hepcidin-25 concentration due to SVR. A comparison of the pre-treatment and post-treatment groups for all 26 patients showed that serum hepcidin-25 levels were significantly lower in the post-treatment group (Fig 3B). To validate our methodology used, several patients’ sera that showed the hepcidin-25 concentration of approximately 5–50 ng/mL were analyzed by a company for clinical laboratory testing. The correlation analysis between the results of the analysis at the laboratory and the results of the measurement in our hand showed a high correlation (R² = 0.9985), demonstrating that the hepcidin-25 measurement data performed in this study are reliable (S7 Fig). Demographic and clinical characteristics of hepatitis C patients with measured serum hepcidin-25 are shown in S1 Table. Our findings suggest that hepcidin expression levels can fluctuate with the loss of viral infection not only during acute inflammation but also during chronic inflammation such as hepatitis C.

Proteolytic cleavage of FPN1 mediated by HCV NS3-4A protease

Hepcidin can bind to the iron transporter FPN1, and hepcidin-FPN1 binding triggers internalization, ubiquitylation, and degradation of FPN1. Therefore, high expression of the hepcidin gene and increased extracellular secretion of hepcidin peptide potentially lead to a reduced steady-state level of FPN1. Flow cytometry analysis of changes in cell surface FPN1 levels upon HCV infection showed that the intensity of FPN1 expression was decreased by HCV infection when FPN1-ectopically expressing cells that were infected with HCV were compared to those without infection (Fig 4A).

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Fig 4. Proteolytic cleavage of FPN1 mediated by HCV NS3-4A protease.

(A) Huh7.5.1 cells with or without HCV infection 1 day after transfection of the FPN1-expression plasmid were harvested at 1 dpi and reacted with anti-FPN1 antibody. The percentage of FPN1-positive cells was counted by flow cytometry. Cells transfected with an EV (nega cont) were used as a negative control. The figure is representative of three experiments with similar results. (B, D) At 1 dpi, cells with or without HCV infection were transfected (B) with the FPN1-expression plasmid together with the NS3-4A expression plasmid or the empty vector, (D) with plasmids expressing FPN1 or FPN1 C326S. At 2 dpt, FPN1, HCV Core, and GAPDH were analyzed by western blotting. (C) FPN1 and GAPDH were analyzed by western blotting with cell lysates transfected with both plasmids expressing FPN1 or FPN1 C326S and Core-NS2, NS3-5B, NS3-4A, or NS3-4A S139A at 2 dpt. (E) HCV-infected cells 3 dpi were immunoprecipitated with anti-FPN1 antibody (BMP033) or rabbit IgG, followed by western blotting using anti-FPN1 antibody (NBP1-21502). HCV Core and GAPDH in input samples were assessed by western blotting without immunoprecipitation. A cell lysate transfected with plasmids expressing FPN1 and NS3-4A was used as a positive control (PC). Endogenous full-length- and processed FPN1 are indicated as an arrowhead and arrow, respectively. (F) MBP-FPN1 (aa 278–292)-sfGFP-FLAG fusion protein as a substrate was reacted with recombinant NS3-4A. After SDS-PAGE and transfer to a PVDF membrane, the processed band stained with CBB (left) was excised, followed by N-terminal analysis. Processing of the substrate fusion protein was confirmed by western blotting using anti-FLAG antibody (right). (G-I) FPN1, HA-tagged NS3-4A, and GAPDH were analyzed by western blotting. Cells were transfected with (F) the plasmid expressing WT- or mutated FPN1 together with the expression plasmid for HA-tagged NS3-4A or EV, (H) the FLAG-FPN1-expressing plasmid together with the HA-NS3-4A expressing plasmid from genotypes 1b, 2a, or 3a, and (I) the plasmids expressing FLAG-FPN1 and HA-NS3-4A in the presence or absence of 1 μM asunaprevir. (B, D, H, I) MG132 and NH₄Cl were added at final concentrations of 10 μM and 10 mM, respectively, 12 h before cell harvesting.

https://doi.org/10.1371/journal.ppat.1011591.g004

To further analyze the effects of HCV infection on the hepcidin-FPN1 system, we performed a sequence motif search that revealed the presence of a consensus cleavage site for HCV NS3-4A protease, D/E-X-X-X-X-C/T↓S/A (where X represents any residue), in the FPN1 protein. To determine whether FPN1 is actually cleaved by the expression of HCV NS3-4A, cells expressing FLAG-tagged FPN1 were generated, and the effect of HCV infection or viral protein expression was analyzed by western blotting. A cleaved form of FPN1 presumably corresponding to the N-terminal side of FPN1 was detected in HCV-infected cells (Fig 4B, lanes 5 and 6) and cells expressing NS3-S4A (Fig 4B, lanes 3 and 4; Fig 4C, lane 4) or NS3-S5B (Fig 4C, lane 3) derived from the viral Con-1 strain (genotype 1b). This band was not observed in cells expressing Core-NS2 or an active center mutant (S139A) of NS3-4A (Fig 4C, lanes 2 and 5). Such FPN1 cleavage by HCV infection was also observed in the case of FPN1 with a mutation in the hepcidin-binding site (C326S) (Fig 4D, lanes 7 and 8), suggesting that the proteolytic cleavage of FPN1 as mediated by HCV NS3-4A is hepcidin independent. The levels of full-length FPN1 and its cleaved form increased at least to some extent by the addition of lysosomal and proteasomal inhibitors (Fig 4B and 4D) and confirmed that the FPN1 cleaved protein produced by HCV NS3-4A expression and that caused by HCV infection are the same molecular size (Fig 4B). Since the anti-FPN1 antibody used in this study recognizes the N-terminal region of FPN1, the C-terminal FPN1 fragment is not detected by western blotting as shown in Fig 4. To detect both the N-terminal and C-terminal fragments of FPN1 cleaved by HCV NS3-4A, we created a new plasmid expressing FLAG-FPN1-myc protein with a FLAG tag at the N-terminus and a c-myc tag at the C-terminus of FPN1. When this FLAG-FPN1-myc was co-expressed with NS3-4A or expressed in HCV-infected cells, the N-terminal fragment of FPN1 was detected with anti-FLAG antibody as the primary antibody, just as it was detected with the anti-FPN1 antibody. In contrast, the C-terminal fragment of FPN1 was detectable with an anti-c-myc antibody (S8 Fig). We confirmed that the observed FPN1 cleavage by HCV NS3-4A was not an artifact of ectopic expression of FPN1 (Fig 4E), because cleaved FPN1 protein not seen in uninfected cells was endogenously detected in HCV-infected cells at 3 days post-infection, and the level of full-length FPN1 decreased accordingly (lane 2 vs. lane 3). The band size of the cleaved form of FPN1 was the same as that observed by co-expression of NS3-4A and FPN1 (lane 4). Due to the limited sensitivity of detection by anti-FPN1 antibodies, immunoprecipitation-WB was performed in this experiment using two different anti-FPN1 antibodies. To identify the cleavage site of FPN1 by HCV NS3-4A, maltose-binding protein (MBP)-FPN1 (aa278-292)-superfolder GFP (sfGFP)-FLAG fusion protein, in which the FPN1 region containing the putative cleavage site was fused with MBP at the N-terminus and sfGFP-FLAG at the C-terminus, was expressed in E. coli and affinity purified. The FPN1-fusion protein obtained was cleaved with a recombinant NS3-4A protease in vitro (Fig 4F). The N-terminal sequence analysis of the cleaved product revealed that the cleavage site by NS3-4A is between Cys (aa284) and Ala (aa285) matched consensus sequence D/E-X-X-X-X-C/T↓S/A in the intracytoplasmic region of the central portion of the FPN1 protein (S9 Fig).

Next, we examined how mutations at the FPN1 cleavage site affect cleavage by NS3-4A. When a mutation was introduced at the P1 site of the cleavage site, both the C284A and C284R mutations were found to maintain cleavage by NS3-4A (Fig 4G, lanes 4 and 6). However, for T283G/C284A double-mutant FPN1, in which both the P1 and P2 sites were replaced, cleavage by NS3-4A was no longer observed (Fig 4G, lane 8). Since the P2 position of the FPN1 cleavage site is Thr (aa283), it is likely that a single mutation to the P1 position at aa284 results in the residue at aa283 becoming P1 and maintaining cleavage by HCV NS3-4A.

In the NS3-4A expression experiment described above, HCV genotype 1b was used; to determine whether FPN1 cleavage activity is also observed in other genotypes of HCV, FPN1 cleavage analysis was performed for NS3-4A from genotypes 2a (JFH-1 strain) and 3a (S52 strain) (Fig 4H, lanes 9 and 11). The results showed that FPN1 is also susceptible to NS3-4A from HCV genotypes 2a and 3a. As expected, FPN1 cleavage by HCV NS3-4A was completely inhibited by the addition of 1 μM of the HCV protease inhibitor asunaprevir (Fig 4I, lanes 11 and 12).

NS3-4A-dependent cleavage of FPN1 in mouse liver tissues

The hepcidin-FPN1 system is also conserved in mice. Human and mouse FPN1 show high amino acid sequence homology, and the sequence around the cleavage site by HCV NS3-4A is completely conserved (S3 Fig). In mouse hepatocellular carcinoma Heplclc7 cells, in which NS3-4A was ectopically expressed, a cleaved form of endogenous FPN1 was observed as expected (Fig 5A, lane 4). To demonstrate that FPN1 can be cleaved by NS3-4A not only in cultured cells but also in liver tissues, a recombinant adenovirus carrying the NS3-4A gene was intravenously injected into mice, and the liver tissues were subjected to western blot analysis. The result showed that the NS3-4A-expressing mice had a cleaved form of FPN1, in good agreement with the results obtained using the cultured cell system (Fig 5A, lane 2).

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Fig 5. Iron store levels in mouse liver tissues and in cultured cells expressing HCV proteins.

(A) Eight-week-old male WT mice were intravenously injected with AdV expressing either GFP or NS3-4A genotype 1b. At 5 dpi, liver tissues were subject to western blotting to detect FPN1 and its cleaved form (red arrow) and HCV NS3 (left). Hepalclc7 cells transfected with the NS3-4A-expressing plasmid or the EV (right). (B) Iron concentrations in cells transfected with the plasmid expressing FPN1 together with the expression plasmid for NS3-4A WT or S139A mutant or EV were measured using the PSAP-method. Mean values obtained from transfectants with the FPN1-plasmid and EV were set as 100%. Results represent the means with SD from three independent measurements. Student’s t test; *P<0.05, **P<0.01. (C) Four groups of mice (N = 4 each) were created in which recombinant adenoviruses were injected as follows: GFP group as a negative control; AdEFLNLGFP, AdEFNCre and AdEFGFP, Core-NS2 group; AdEFLNLHCVCore/NS2 and AdEFNCre, NS3-4A group; AdEFHCVNS3/4A, Core-NS2 and NS3-4A group; AdEFLNLHCVCore/NS2, AdEFNCre and AdEFHCVNS3/4A. Thin section slices were prepared from livers at 5 dpi and stained with Berlin blue. Representative images of two mouse livers from each group are shown. (D) Elemental analysis was performed with the NanoSuit method at the six spots positive for staining (Blue staining positive) and unstained parts (Background) in slices from the Core-NS2 group described in S13 Fig. (Left) Elemental composition graphs of energy (keV) vs. cps per eV are shown. Areas including the Fe peaks in the sample positive for blue staining and the corresponding area in the background sample are expanded. (Right) The weight concentrations of Fe measured were quantified and are expressed as weight percentages (wt%). Student’s t test; **P<0.01. (E) Total iron scores of 10 fields of view in the livers from the four groups described in (C) were individually evaluated by means of histological hepatic iron index. Results represent the plots separately for each group. Welch’s t test; *P<0.05, **P<0.01, ***P<0.001, ns P>0.05.

https://doi.org/10.1371/journal.ppat.1011591.g005

Iron deposition in the liver tissues of mice expressing HCV proteins

Next, we used the Nitroso-PSAP method to analyze whether intracellular iron levels were altered by HCV NS3-4A and/or FPN1 expression: intracellular iron levels were decreased in cells with FPN1 overexpression compared to control cells transfected with an empty vector (S10 Fig, bars 1 and 3). In both cell types, HCV infection led to increases in cellular iron levels (S10 Fig, bars 2 and 4). Ectopic expression of HCV NS3-4A (wild type) increased iron levels in cells, whereas expression of the inactive NS3-4A mutant (S139A) did not; instead, NS3-4A S139A-expressing cells showed lower iron levels than in control cells (Fig 5B).

Finally, to analyze the iron deposition in liver tissues from HCV protein-expressing mice, we injected HCV Core-NS2-expressing AdV or NS3-4A-expressing AdV alone, or Core-NS2-expressing AdV and NS3-4A-expressing AdV together, and 5 days later analyzed liver tissues with Berlin blue staining. As expected, FPN1 cleavage was observed in the livers of NS3-4A-expressing mice (S11 Fig), and hepcidin and BMP6 mRNA expression levels were significantly higher in Core-NS2-expressing mice than in NS3-4A-expressing mice alone or negative control, GFP-expressing mice (S12 Fig). No positive staining with Berlin blue was observed in the negative control mice, but a few positive images were observed in mice expressing HCV Core-NS2 alone or NS3-4A alone. clustered Berlin blue-positive regions in the liver (Fig 5C and 5E). Independently of the experiment, we compared iron deposition in the liver of HCV Core-NS2-expressing mice versus core-NS2- and NS3-4A co-expressing mice and confirmed that Core-NS2 and NS3-4A co-expressing mice showed significantly higher iron deposition (S13 Fig). Since the HCV core-NS2-expressing liver tissues showed Berlin blue positivity but to a limited extent, trace elemental analysis was performed by Scanning Electron Microscopy/Energy Dispersive X-Ray Spectroscopy (SEM/EDS) combined with NanoSuit-Correlative Light and Electron Microscopy (CLEM) to verify whether iron was actually deposited in the stain-positive areas. The Berlin blue-positive areas clearly showed more iron-specific characteristic X-rays and higher mass percentages of iron compared with the background areas in liver tissues (Fig 5D). Based on the Berlin blue staining data, the iron deposition levels in the liver tissues of each mouse group were compared by total iron score (Fig 5E). Both HCV protein-expressing mouse groups showed significantly higher scores than the negative control group. HCV Core-NS2 and NS3-4A co-expressing mouse groups showed higher iron scores than the Core-NS2 expressing mouse group.

Together with other findings obtained in this study, we conclude that both transcriptional upregulation of hepcidin gene expression and proteolytic cleavage of FPN1 are involved in the HCV-induced increase in intracellular iron levels.

Ethics statement

The experiments using clinical samples were approved by the Ethics Committee of Hamamatsu University School of Medicine. Informed consent in writing was obtained from each patient. Animal husbandry procedures and experiments using WT and Creb3l3^−/− mice were performed in accordance with the Regulations for Animal Experiments of Hamamatsu University School of Medicine and were approved by the Animal Experiment Committee of the university.

Reagents

Human hepcidin-25 peptide and MG-132 (Peptide Institute, Osaka, Japan) and HCV NS3 protease inhibitor, asunaprevir (BMS-650032) were used.

Cell culture

The mouse hepatoma line Hepa1c1c7, human hepatoma HepG2 cells, and HuH-7 and its derivative cell line Huh7.5.1, which is HCV negative and highly permissive for HCV infection [47] (a gift from Francis V. Chisari, The Scripps Research Institute, La Jolla, CA, USA) were grown essentially as described [48]. To arrest proliferation of hepatoma cells and allow the culture to continue for several weeks without passaging, Huh7.5.1 cells were cultured in Dulbecco’s modified Eagle medium containing 1% dimethyl sulfoxide (DMSO) [19] for 14 days before being used for HCV infection [20]. CREBH-KO cells derived from Huh7.5.1 cells were produced using the CRISPR-Cas9 system as described [22].

Plasmids

pcDNA-FPN1, which expresses full-length FPN1 (1,716 amino acids), was constructed by inserting PCR products of corresponding FPN1 regions, which were amplified with Huh7.5.1-derived cDNA as a template, into pcDNA3.1 (Thermo Fisher Scientific, Waltham, MA, USA). To make expression plasmids for mutated FPN1 with substitutions and partial deletions, the GENEART Site-Directed Mutagenesis System (Thermo Fisher Scientific) and the Dpn I method (New England Biolabs, Ipswich, MA, USA), respectively, were used. To construct pcDNA-FLAG-FPN1, a PCR product obtained with a sense primer designed to contain the FLAG tag at the N-terminus of FPN1 and pcDNA-FPN1 as a template was inserted into pcDNA3.1. To create pcDNA-hepcidin, which is a hepcidin-expressing plasmid, a PCR product corresponding to the cDNA of the entire preprohepcidin region, which was amplified as per above, was inserted into pcDNA3.1. To construct promoter reporters for hepcidin, 2.7 kb region upstream of the translation initiation site of the hepcidin gene was obtained by PCR and inserted into pGL4.10 (Promega, Madison, WI, USA), yielding pGL-hepcidin pr. The BMP6 promoter reporter pGL-BMP6pr was constructed by inserting the BMP6 promoter sequence derived from pKM2L-phBMP6 (RDB05474), which was provided by RIKEN BRC through the National Bio Resource Project of MEXT, Japan, into pGL4.10. Expression plasmids for the HCV proteins pCAG-Core-NS2 were previously described [49].

pCAG-HACon1NS3NS4A, which expresses full-length NS3 and NS4A (derived from HCV genotype 1b, Con-1 strain) with an HA tag at the N-terminus of NS3, and its mutated construct pCAG-HACon1NS3NS4A S139A that has a point mutation of serine to alanine at position 139, were previously described [33]. To construct pCAG-HAJFH1NS3NS4A and pCAG-HAS52NS3NS4A, PCR fragments encoding the NS3-NS4A region derived from the JFH-1 strain (genotype 2a) and S52 strain (genotype 3a) [50], respectively, were digested with XhoI-NotI, followed by replacement with the corresponding part of pCAG-HACon1NS3NS4A. To construct pCAG-HACon1NS3NS5B, PCR fragments encoding the NS3-NS5B region derived from the Con-1 strain were replaced with the corresponding part of pCAG-HACon1NS3NS4A using In-Fusion HD Cloning Kit (Takara Bio Inc., Shiga, Japan).

Virus infection and DNA transfection

HCV stock derived from J6/JFH-1 was prepared as previously described [51]. Naive Huh7.5.1 cells were infected with HCV J6/JFH-1 at various MOI for 4 h. Lipofectamine LTX (Thermo Fisher Scientific) was used for DNA transfection.

RNA extraction and RT-PCR

Total cellular RNAs isolated by the TRI reagent (Molecular Research Center, Cincinnati, OH, USA) were transcribed using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). Aliquots of cDNAs were subjected to 40 cycles of PCR amplification. Primer sequences used are shown in S2 Table. Quantitative (q)PCR was performed with the CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA) using THUNDERBIRD SYBR qPCR mix (Toyobo, Osaka, Japan). Briefly, reverse-transcribed cDNAs together with 6 pmol of forward and reverse primers were used for PCR. The thermal cycling conditions were: 1 min at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 30 s. The RNA expression data were normalized to mRNA levels of β-actin or GAPDH using the comparative threshold cycle method.

Luciferase reporter assay

Cells seeded in a 24-well plate at a density of 5 × 10⁴cells/well were transiently co-transfected with Renilla luciferase and firefly luciferase vectors with or without HCV infection. For conditions with HCV infection, the hepcidin- or BMP6-promoter reporter vectors were introduced into the cells at 1 dpi of Huh7.5.1 cells (MOI = 0.5). After 2 days, luciferase activities were measured using the Dual-Luciferase reporter assay system (Promega) and a Synergy H1 Hybrid Multi-Mode Microplate Reader (Biotek, Winooski, VT, USA). Each firefly luciferase activity was normalized to the Renilla luciferase activity expressed from pGL4.73 or pGL4.74 (Promega).

Flow cytometry analysis

Huh7.5.1 cells transfected with pcDNA-FPN1 with or without HCV infection were collected with a scraper, followed by centrifuging at 500 × g for 3 min. The cells were incubated with the primary antibody (anti-SLC40A1 (ab85370)) for 1 h. After washing three times with PBS, the cells were incubated with a secondary antibody (anti-rabbit Alexa 488) for 1 h. Samples were measured with a Gallios Flow Cytometer (Beckman Coulter, Inc., Brea, CA, USA) and analyzed with FlowJo FCM analysis software.

Immunoprecipitation and western blotting

Cells were washed with PBS and lysed for 5 min on ice in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS) with a protease inhibitor cocktail (Roche Diagnostics, Rotkreuz, ZG, Switzerland). Lysates were cleared by centrifugation, and protein concentrations were determined using the BCA Protein Assay Reagent (Thermo Fisher Scientific). For tissue samples, liver tissues were homogenized with RIPA buffer with a protease inhibitor cocktail. Immunoprecipitation was performed by incubating cell lysates for 2 h at 4°C with the appropriate antibody and then for 2 h at 4°C with Protein G Mag Sepharose (Cytiva, Tokyo, Japan). Immunoprecipitates were washed three times in RIPA buffer and subjected to SDS-PAGE. Samples were treated with a 3-fold volume of 10 M urea at 37°C for 15 min prior to SDS-PAGE. Following SDS-PAGE of samples with equal protein contents, the proteins on the gels were transferred to a polyvinylidene difluoride membrane. After blocking for 1 h, the membranes were incubated with first antibodies for 1 h at room temperature. After washing, signals were detected using horseradish peroxidase-conjugated antibody (Cell Signaling Technology, MA) and chemiluminescence (GE Healthcare Japan, Tokyo, Japan). The antibodies used in this study are listed in S3 Table.

Chromatin immunoprecipitation

The ChIP assay was performed with the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s protocol. Nuclear extracts were immunoprecipitated with a polyclonal anti-CREBH antibody (Affinity Biosciences, Cincinnati, OH, USA), monoclonal anit-Smad1 antibody (Cell Signaling Technology), or normal rabbit IgG (Cell Signaling Technology) as a negative control. DNA samples prepared from the precipitates were subjected to qPCR using primers encompassing the human hepcidin and BMP6 promoter as shown in S2 Table.

Metallo assay

Cells seeded in a 12-well plate at a density of 1 × 10⁵ cells/well were transiently co-transfected with pcDNA-Fpn1 and pCAG-HANS3NS4A or -NS3NS4A S139A. After 2 days, intracellular iron ions (Fe²⁺ and Fe³⁺) in cell lysates were measured with the Metallo Assay Iron Assay Kit LS Nitroso-PSAP method (Metallogenics, Chiba, Japan). For HCV infection experiments, cells were infected with HCV J6/JFH-1 at various MOI for 4 h, and intracellular iron ions were measured at 3 dpi.

Detection of Fe²⁺ using the Labile Ferrous Ion Detecting Probe FeRhoNox-1

Intracellular iron Fe²⁺ ions via orange (red) fluorescence after treatment with the activatable fluorescent probe FeRhoNox-1 (GORYO Chemical, Hokkaido, Japan) were specifically observed with a laser scanning microscope, FV-1000 (OLYMPUS, Tokyo, Japan), for confocal microscopic analysis. Cytoplasmic luminescence intensity was measured using Image J software 1.47v (National Institutes of Health, Bethesda, MD, USA).

Quantification of Hepcidin-25 by LC-MS/MS analysis

Synthetic human hepcidin (DTHFPICIFCCGCCHRSKCGMCCKT) and stable isotope-labeled (SI) human hepcidin (DTH[¹³C₉, ¹⁵N]FPICI[¹³C ₉, ¹⁵N]FCC[¹⁵N]GCCHRSKCGMCCKT) as an internal standard (IS) were purchased from the Peptide Institute. The hepcidin and IS stock solutions (100 μg/mL) were generated by diluting 50/50/0.1 (v/v/v) with water/ethanol/formic acid and were stored at −20°C in Protein LoBind Tubes (Eppendorf, Hamburg, Germany). Working solutions of hepcidin and IS were prepared by diluting stock solutions at 2 μg/mL and 1 μg/mL in 50/50/0.1 (v/v/v) acetonitrile (ACN)/water/formic acid, respectively. Standard samples were obtained by serial dilutions of the working solution.

For measuring hepcidin concentrations in culture supernatants of HCV-infected cells, equal amounts of 4% TCA and internal standard working solution were added to 10 mL of HCV-infected culture supernatants and centrifuged at 9,500 x g for 25 min. After removing the supernatants, the precipitates were dissolved in 200 μL of 40/60/1 (v/v/v) ACN/water/formic acid and filtered through a 0.20-μm polytetrafluoroethylene (PTFE) membrane.

To determine the hepcidin level in hepatitis C patient sera, sample preparations from sera were performed using Oasis HLB cartridges (Waters, Milford, MA, USA). The manufacturer’s protocol was optimized as follows. Briefly, 200 μL human serum was diluted v/v with 0.1% formic acid solution after adding the internal standard working solution. After a vortex step for 10 s and centrifugation, supernatants were entirely transferred into the Oasis HLB, which was pretreated with 1 mL of methanol and 1 mL of water under vacuum. The cartridges were washed sequentially with 1 mL of 75/30/5 (v/v/v) water/methanol/5% ammonium hydroxide. The extracts were eluted by 1 mL of 10/90/0.1 (v/v/v) water/methanol/formic acid and evaporated at 43°C for 3 h with a Savant SpeedVac Plus SC110A concentrator (SAVANT Instruments, Holbrook, NY, USA). After evaporation, samples were dissolved in 200 μL of 40/59/1 (v/v/v) ACN/water/formic acid and filtered through a 0.20-μm PTFE.

The LC system consisted of an Acquity ultra performance liquid chromatography (UPLC) unit (Waters). The mobile phases were 0.1% formic acid in water (A) and 0.1% formic acid in ACN (B), and the flow rate was 0.3 mL/min. After injection, mobile phase B was linearly ramped from 15% to 35% in 5 min, then reached 100% in 2 min. The proportion of B was maintained at 100% for 3 min and was then reduced rapidly to 15% for 0.1 min for re-equilibrating. The run duration of each sample lasted 18 min. The MS/MS analysis was performed in positive ESI mode using a 4000Q-TRAP quadropole linear ion-trap hybrid mass spectrometer (AB SCIEX, Tokyo, Japan) with the following settings: ion spray voltage, 4500 V and temperature, 600°C. Hepcidin was detected by multiple reaction monitoring. The ions were selected in the first quadrupole (Q1) and collided with nitrogen gas in the second quadrupole (Q2), and the product ions were detected in the third quadrupole (Q3). Human hepcidin was quantified by using the ion transition 698.3 (Q1) → 109.9 (Q3) for the non-labeled hepcidin and 563.0 (Q1) → 69.9 (Q3) for IS.

Recombinant adenoviruses

The cDNA encoding HCV Core-NS2 derived from the JFH-1 strain was cloned into the cosmid cassette of pxAdEFLNLw-FVF containing the full-length AdV genome. The Cre recombinase inducible AdV (AdEFLNLHCVCore/NS2) and Cre-expressing AdV (AdEFNCre) were prepared as previously described [52]. The AdV vector AxEFLNLG as a control of Cre efficiency and the purification method were as previously described [53]. To produce the AdV vector expressing HCV NS3-4A (AdFEHCVNS3/4A), the cDNA encoding the HCV NS3-NS4A region of JFH-1 was cloned into the cosmid cassette of pAxEFwit2 [54], which contains the EF1alpha promoter and the full-length AdV genome excluding E1 and E3 coding regions. AdFEHCVNS3/4A and AdV expressing GFP (AdEFGFP) as a control were prepared [54].

Mouse experiments

Male C57/BL6J (WT) mice were obtained from SLC Japan (Hamamatsu, Japan), and the CREBH null-mutant, Creb3l3tm1.1Sad/J (Creb3l3^−/−) mice were a kind gift from Prof. Hitoshi Shimano, Faculty of Medicine, University of Tsukuba, Japan [55]. For AdV infection, eight-week-old male mice were infected with AdEFNCre [53] together either with AdEFGFP or AdEFLNLHCVCore/NS2 at 1.0 × 10⁹ pfu per mouse, followed by collecting liver samples 5 dpi. WT mice were infected either with AdEFGFP or AdFEhHCVNS3/4A at 2.0 × 10⁹ pfu per mouse, followed by collecting liver samples at 5 dpi. For co-expression of HCV C-NS2 and NS3-4A, eight-week-old male mice were infected with AdEFNCre and AdEFLNLHCVCore/NS2 at 1.0 × 10⁹ pfu per mouse, together with AdFEHCVNS3/4A at 2.0 × 10⁹ pfu per mouse, followed by collecting liver samples at 5 dpi. Iron deposits were assessed by scoring according to their size and their cellular and lobular locations in hepatic lobules. Total iron scores were calculated from the sum of hepatocytic, sinusoidal, and portal iron scores as previously described [56].

NanoSuit-CLEM method combined with SEM-EDS

The preparation of specimens for observation with the NanoSuit method was previously described [57]. Spots stained with Berlin blue and unstained parts on the slides of mouse liver tissues were investigated by SEM observation using CLEM-EDS and the NanoSuit method as previously described [58,59]. Elemental analysis of iron was performed with Low-vacuum SEM (Lv-SEM) (TM4000Plus; HITACHI, Tokyo, Japan; accelerating voltage: 15 kV) equipped with an EDS (X-stream-2; Oxford instruments, Oxford, UK). AZtecOne software (Oxford Instruments) was used for the EDS analysis. The weight concentrations of detected elements were quantified and expressed as weight percentages (wt %), which shows the relative concentration of an element in the analyzed area.

Statistical analyses

All statistical analyses were conducted using IBM SPSS Statistics version 25 (IBM, Armonk, NY, USA). Values are expressed as means with standard deviation (SD). Statistical significance was calculated using an unpaired Student’s t test, Welch’s t test or Mann-Whitney rank sum test. Statistical significance was set at P<0.05.