Association of Hepatitis C Virus Replication with the Catecholamine Biosynthetic Pathway

A bidirectional negative relationship between Hepatitis C virus (HCV) replication and gene expression of the catecholamine biosynthetic enzyme L-Dopa decarboxylase (DDC) was previously shown in the liver and attributed at least to an association of DDC with phosphatidylinositol 3-kinase (PI3K). Here, we report that the biosynthesis and uptake of catecholamines restrict HCV replication in hepatocytes, while HCV has developed ways to reduce catecholamine production. By employing gene silencing, chemical inhibition or induction of the catecholamine biosynthetic and metabolic enzymes and transporters, and by applying the substrates or the products of the respective enzymes, we unravel the role of the different steps of the pathway in viral infection. We also provide evidence that the effect of catecholamines on HCV is strongly related with oxidative stress that is generated by their autoxidation in the cytosol, while antioxidants or treatments that lower cytosolic catecholamine levels positively affect the virus. To counteract the effect of catecholamines, HCV, apart from the already reported effects on DDC, causes the down-regulation of tyrosine hydroxylase that encodes the rate-limiting enzyme of catecholamine biosynthesis and suppresses dopamine beta-hydroxylase mRNA and protein amounts, while increasing the catecholamine degradation enzyme monoamine oxidase. Moreover, the NS4B viral protein is implicated in the effect of HCV on the ratio of the ~50 kDa DDC monomer and a ~120 kDa DDC complex, while the NS5A protein has a negative effect on total DDC protein levels.


Introduction
Hepatitis C virus (HCV) infection, with~71 million cases worldwide, causes chronic liver injury that could eventually lead to liver cirrhosis and hepatocellular carcinoma (HCC) [1]. The global epidemic of HCV is distributed unevenly, with the Eastern Mediterranean region and Europe having the highest prevalence [1][2][3]. The HCV genome, a positive sense, single-stranded RNA, encodes a single polyprotein of~3000 amino acids [4,5]. This is proteolytically cleaved into structural proteins (core, E1 and E2) that form the viral capsid, p7, which is necessary for the assembly and release of infectious virions, and non-structural

In Vitro Transcription
Ten micrograms of viral constructs were linearized with MluI and were used for in vitro transcription as described previously [56]. In brief, plasmid DNA was extracted with phenol and chloroform, precipitated with ethanol and dissolved in RNase-free water. In vitro transcription was performed in 80 mM HEPES (pH 7.5) containing 12 mM MgCl2, Viruses 2021, 13, 2139 4 of 26 2 mM spermidine, 40 mM dithiothreitol (DTT), 3.125 mM of each nucleoside triphosphate, 1 U/µL of RNasin (Promega Corporation, Madison, WI, USA), 0.1 µg plasmid DNA/µL, and 0.6 U/µL of T7 RNA polymerase (Promega). Afterwards, 1.2 U of RNase-free DNase (Promega) per µg of plasmid DNA was added to terminate transcription. The viral RNA was extracted with acidic phenol and chloroform, precipitated with isopropanol, and dissolved in RNase-free water.

Transfection Assays
For viral protein overexpression, Huh7-Lunet cells were electroporated with 60 µg of plasmids pEGFP-NS4B, pcDNA3-NS5A, or the empty vector pcDNA3 (In vitro gen). In DDC silencing experiments, Huh7.5 cells were electroporated with 60 µg of the shDDC or shControl expressing plasmids. 4 × 10 6 cells were detached with trypsin, washed with PBS 1×, resuspended in Cytomix [57] containing 2 mM ATP and 5 mM glutathione, mixed with plasmid DNA and electroporated at 220 V, 975 µF with a Gene Pulser system (Bio-Rad, Hercules, CA, USA). The cells were immediately transferred to 20 mL of complete DMEM and subsequently seeded as required for the assay.

Preparation and Titration of Virus Stocks and Infection Assays
HCV virus stocks were generated in Huh7-Lunet cells as described elsewhere [58]. In brief, virus stocks were generated by electroporation of in vitro transcribed viral RNAs, and the supernatants of transfected cells were harvested 24, 48, 72, and 96 h post-transfection (p.t). After filtration of the supernatants through 0.45-µm-pore-size filters, the virus particles were concentrated by ultracentrifugation through a self-generating iodixanol gradient. The collected fractions containing virus particles were further concentrated using Centricon Plus-70 centrifugal filter devices (Merck-Millipore, Burlington, MA, USA) according to the manufacturer's instructions. HCV was titrated as described elsewhere [59], using the JFH1 NS5A-specific mouse monoclonal antibody 9E10 [59]. Infectivity titers were expressed as the 50% tissue culture infective dose (TCID50)/mL. Virus stocks were used to infect naive Huh7.5 cells. The culture medium was exchanged 4 h post-virus inoculation.

Measurement of Intracellular ATP Levels
Intracellular ATP was measured using the ViaLight HS BioAssay kit (Lonza, Basel, Switzerland) according to the manufacturer's protocol, in a GloMax 20/20 single-tube luminometer (Promega Corporation, Madison, WI, USA) for 1 s, as recommended by the manufacturer. ATP levels were normalized to total protein amounts.

RNA Quantification by Reverse Transcription-Quantitative PCR (RT-qPCR)
Total RNA extraction from cells was performed using nucleoZOL reagent (Macherey-Nagel, Duren, Germany) according to the manufacturer's instructions. cDNA synthesis was carried out with Moloney murine leukemia virus reverse transcriptase (Promega Corporation, Madison, WI, USA) based on the manufacturer's protocol. For HCV positivestrand RNA quantitation, reverse transcription (RT) reactions included the HCV-specific primer JFH1-354R, as well as the primer YWHAZ-R (Table 1), specific for the housekeeping gene 14-3-3-zeta polypeptide (YWHAZ) used as internal control (3.5 pmol/µL of each primer). For the quantification of cellular transcripts, oligo(dT) primers (New England Biolabs, Ipswich, MA, USA) were included. Real-time quantitative PCR was performed using Luna ® Universal qPCR Master Mix (New England Biolabs, Inc. Ipswich, MA, USA), as well as primer pairs specific for the HCV IRES (JFH1-276F and JFH1-354R), the exons 10-12 of full-length DDC mRNAs, TH, DBH, MAO-A, MAO-B, VMAT2, OCT1, NRF2, HO-1 and VEGFA mRNAs. The YWHAZ housekeeping gene was used as a normalization control in all qPCR reactions, as its expression was not affected upon viral infection.

Indirect Immunofluorescence
Indirect immunofluorescence analysis of human DDC and HCV NS5A in Huh7-Lunet cells was performed as previously described [7]. Cells were seeded onto glass coverslips in 24-well plates (5 × 10 4 cells/well). At 48 h post-electroporation with plasmid DNA, cells were fixed with 3% paraformaldehyde for 10 min at room temperature and permeabilized by incubation in PBS supplemented with 0.5% Triton X-100 for 5 min. Staining of DDC was performed by using an anti-DDC C-T polyclonal antibody at a dilution of 1:20, while staining of HCV NS5A was performed by using the mouse monoclonal antibody 9E10 [59] at a dilution of 1:1000. Bound primary antibodies were detected by using goat anti-mouse antibodies conjugated to Alexa-Fluor 488, or goat anti-rabbit antibodies conjugated to Alexa-Fluor 546 at a dilution of 1:1000. DNA was stained with Hoechst 33258 (Thermo Fisher Scientific). HCV NS4B was detected through GFP fluorescence. Images were acquired with the Leica TCS-SP8 confocal microscope. Fluorescence quantitation and colocalization analysis was carried out using Icy software [61,62]. Pearson's correlation coefficient and Manders' colocalization coefficients were calculated using Colocalization Studio plugin.

ELISA Assay
Dopamine was quantified in cell supernatants using the Dopamine ELISA kit (IBL International, Hamburg, Germany), according to the manufacturer's instructions. To the supernatants collected after treatment, 30 µM EDTA and 110 µM L-ascorbic acid were added to prevent dopamine oxidation, and subsequently samples were centrifuged at 2000 g for 20 min at 4 • C for removal of cell debris.

Statistical Analysis
In all diagrams, bars denote mean values of at least 3 independent experiments in triplicate and error bars standard deviation. Statistical analyses were performed using Student's t-test and results with p < 0.05 were considered as statistically significant. Calculations were carried out using Excel Microsoft Office ® (Microsoft Corporation, Redmond, WA, USA) or Prism (GraphPad Software, Inc., San Diego, CA, USA).

Ethics Statement
This material is the authors' own original work, which has not been published in whole or in part elsewhere. All authors have been personally and actively involved in substantive work leading to the manuscript, and will hold themselves responsible for its content. This study does not involve humans or animals.

Silencing of L-Dopa Decarboxylase (DDC) Increases HCV Replication and Suppresses the Antiviral Effect of DDC Substrates
Having previously identified DDC as a novel cellular factor regulated by HCV infection and that in turn DDC overexpression/chemical inhibition affects viral RNA replication [7], hereby we performed gene silencing to confirm the negative effect of DDC on HCV replication and to examine whether this effect is mediated by the biosynthetic role of DDC. To silence DDC, human hepatoma Huh7.5 cells were electroporated with an shDDC plasmid vector or a scramble negative control shRNA plasmid (shControl) and subsequently infected with Jc1 or the reporter JcR2A virus. The silencing of DDC Viruses 2021, 13, 2139 7 of 26 was shown to positively affect HCV proliferation with a significant increase up to~3-fold observed at the level of Jc1 NS5A protein ( Figure 1A), viral positive-strand RNA ( Figure 1B) and JcR2A replication-derived Renilla luciferase (R-Luc) activity ( Figure 1C) in cell lysates (see also Figure S1A,B; replication kinetics of the viruses). DDC silencing was confirmed by measuring intracellular DDC mRNA ( Figure S1C) and protein levels ( Figure 1A). Next, we confirmed that the favorable role of DDC silencing on HCV is exerted at the level of viral RNA replication by introducing the subgenomic JFH1 replicon in stable transfected cell lines expressing shDDC or shControl RNA ( Figure S1D,E).
Having previously identified DDC as a novel cellular factor regulated by HCV infection and that in turn DDC overexpression/chemical inhibition affects viral RNA replication [7], hereby we performed gene silencing to confirm the negative effect of DDC on HCV replication and to examine whether this effect is mediated by the biosynthetic role of DDC. To silence DDC, human hepatoma Huh7.5 cells were electroporated with an shDDC plasmid vector or a scramble negative control shRNA plasmid (shControl) and subsequently infected with Jc1 or the reporter JcR2A virus. The silencing of DDC was shown to positively affect HCV proliferation with a significant increase up to ~3-fold observed at the level of Jc1 NS5A protein ( Figure 1A), viral positive-strand RNA ( Figure 1B) and JcR2A replication-derived Renilla luciferase (R-Luc) activity ( Figure 1C) in cell lysates (see also Figure S1A,B; replication kinetics of the viruses). DDC silencing was confirmed by measuring intracellular DDC mRNA ( Figure S1C) and protein levels ( Figure 1A). Next, we confirmed that the favorable role of DDC silencing on HCV is exerted at the level of viral RNA replication by introducing the subgenomic JFH1 replicon in stable transfected cell lines expressing shDDC or shControl RNA ( Figure S1D,E).  Moreover, DDC silencing attenuates the negative impact of DDC enzymatic substrates on JcR2A replication ( Figure 1D). Specifically, non-cytotoxic concentrations of L-Dopa and 5-HTP ( Figure S2A,B) reduced virus-derived R-Luc activity in shControl-electroporated cells by 50% and 57%, respectively ( Figure 1D), in consistence with our previous data [7]. This reduction was confirmed for Jc1 RNA and protein ( Figure S2C,D). However, a significantly lower effect was observed for L-Dopa (15%) and 5-HTP (39%) ( Figure 1D) in shDDCelectroporated cells. This suggests that DDC affects HCV replication at least through the processing of L-Dopa and 5-HTP.

Exogenous Application of the DDC Protein Products Dopamine and Serotonin Decreases HCV Replication
We then examined whether the virus is affected by the exogenous addition of DDC products dopamine (DA) and serotonin (5-HT). Treatment of JcR2A-infected Huh7.5 cells with DA or 5-HT for 72 h starting from 4 h post-virus inoculation, reduced virus-derived R-Luc activity by~2-fold ( Figure 2A, see also Figure S3A,B for cytotoxicity profile). Accordingly, lower viral RNA ( Figure 2B) and protein ( Figure 2C) levels were detected in DA-treated cells as compared to mock-treated (Control) ones. The negative effect of DA on viral RNA replication was confirmed in the HCV genotype 1b (Con1) subgenomic replicon cell line ( Figure 2D). DA did not significantly alter DDC mRNA and protein levels in both infected and mock-infected cells ( Figures 2C and S4A). To discriminate if DA negatively influences HCV through the dopaminergic receptor signaling, we applied prochlorperazine (PCZ), an antagonist of the D2 dopamine receptor, to JcR2A-infected cells. We selected to study D2 as it has been detected and well-characterized in the liver [63][64][65]. PCZ had no impact on viral replication either in the absence or presence of exogenous dopamine ( Figure 2E). The above data combined suggest that the effect of dopamine on HCV is possibly exerted by its intracellular production and uptake and not through dopamine receptor signaling.

HCV Replication Is Enhanced by the Use of an Inhibitor of the Monoamine Transporter VMAT2
To directly address the importance of catecholamines and serotonin uptake and storage on HCV replication, we inhibited the activity of VMAT2. VMAT2 is responsible for the uptake of catecholamines and serotonin from extracellular medium through up-regulation of their transporters, and for their transfer in storage vesicles, where DA is converted to NE by the enzyme DBH [66][67][68][69]. Specifically, Huh7.5 cells mock-infected or infected with the reporter JcR2A or the Jc1 virus were subsequently treated with non-cytotoxic concentrations of the VMAT-specific irreversible inhibitor reserpine ( Figure S3D) [70,71]. Reserpine has been shown to deplete intracellular catecholamine stores [70,71] by inhibiting dopamine and norepinephrine transporter expression [72,73] and activity [69] and thus the uptake of catecholamines, through a VMAT-and VMAT-containing catecholamine storage vesicles-dependent mechanism [66][67][68][69]. In agreement with the above, reserpine reduced the levels of DBH protein in Huh7.5 cells ( Figure 3C), possibly due to the inhibition of dopamine import in the cytoplasmic vesicles.  Error bars indicate standard deviations. * p < 0.01, ** p < 0.001 vs. Control. [70,71]. Reserpine has been shown to deplete intracellular catecholamine stores [70,71] by inhibiting dopamine and norepinephrine transporter expression [72,73] and activity [69] and thus the uptake of catecholamines, through a VMAT-and VMAT-containing catecholamine storage vesicles-dependent mechanism [66][67][68][69]. In agreement with the above, reserpine reduced the levels of DBH protein in Huh7.5 cells ( Figure 3C), possibly due to the inhibition of dopamine import in the cytoplasmic vesicles.  Reserpine enhanced the replication of HCV as the JcR2A replication-derived R-Luc activity was increased by up to 2-fold ( Figure 3A). Similarly, Jc1 RNA and protein levels were higher in reserpine-treated cells, as compared to mock-treated (Control) cells ( Figure 3B,C). A comparable up-regulation was observed in the subgenomic replicon system ( Figure S5A). VMAT2 protein expression was confirmed in Huh7.5 cells ( Figure 3C), in agreement to its previously reported detection in hepatocytes. These data probably suggest that VMAT2 negatively correlates with HCV replication. Moreover, in reserpine-treated cells the ex-tracellular dopamine levels were 2.25-fold higher than in control (mock-treated, Control) cells, as determined by ELISA assay in cell supernatants ( Figure 3D), and similar to the dopamine levels in a plain culture medium not incubated with cells (Medium). Thus, cells appeared to uptake/metabolize the medium-containing dopamine in agreement with previous studies [74], while reserpine seemed to completely abrogate this process. On the other hand, HCV infection did not cause any significant change in the extracellular dopamine levels ( Figure 3D, Jc1 Control). To further address the effect of reserpine on the uptake of dopamine by the cell, we examined the expression of OCT1, a major catecholamine transporter in hepatocytes [25,26]. Indeed, reserpine reduced OCT1 mRNA levels, as determined by qPCR in the cell lysates of both infected and mock-infected Huh7.5 cells ( Figure 3E). The above data combined suggest that reserpine may increase viral replication through a reduction of OCT1 transporters expression that lowers the intracellular levels of catecholamines. This premise is substantiated by the finding that reserpine abolished the negative effect of exogenously supplied dopamine on viral replication ( Figure 3F). Finally, a reserpine-mediated HCV-replication increase occurred concomitantly with a reduction in DDC mRNA levels ( Figure S4B), which was expected based on the negative correlation between viral replication and DDC expression reported in our previous [7] and current studies. In mock-infected cells, DDC expression was not altered by reserpine.

The Role of DBH, the Enzyme Catalyzing the Conversion of Dopamine to Norepinephrine, for HCV Replication
Treatment of HCV-infected cells with non-cytotoxic concentrations of the DBH-specific inhibitor nepicastat ( Figure S3E), which inhibits the biosynthesis of norepinephrine (NE) [75], did not seem to affect either HCV replication-derived luciferase activity or viral RNA and protein levels ( Figures 4A-C and S5B). On the other hand, externally provided NE reduced viral replication in infected cells ( Figure 4D,E), despite that NE-treatment enhanced the intracellular ATP content ( Figure S3F), possibly through up-regulation of aerobic glycolysis [31,76]. Moreover, treatment with reserpine that has been previously reported to inhibit the uptake of NE [69,72] alleviated the negative effect of NE on viral replication ( Figure S5C), similarly to our observation for dopamine and reserpine co-treatment ( Figure 3F). Finally, NE reduced DDC mRNA levels ( Figure S4C), which was also reverted by the co-treatment with reserpine ( Figure S4D). The above results suggest that when NE is restricted inside the cytosolic vesicles, it cannot influence HCV replication, whereas its uptake and accumulation in the cell cytosol leads to the inhibition of viral replication.

Inhibition of MAO Monoamine Degradation Enzymes Reduces HCV Replication
Thus far, our data suggested that the accumulation of catecholamines in the cell cytosol negatively affected HCV replication. Part of the accumulated cytosolic-located catecholamines can either undergo auto-oxidation, producing toxic quinones that can also generate ROS [77], or can be deaminated by MAO enzymes. To examine if MAO function could contribute to the regulation of HCV replication, we performed an inhibition of MAO-A and MAO-B in Huh7.5 cells. MAO-A has a higher affinity to 5-HT, while both MAO isoforms metabolize catecholamines [20] and their inhibition causes the accumulation of these monoamines in the cytoplasm. We observed that virus replication was up to 2-fold lower in the presence of the MAO-A and MAO-B irreversible inhibitor phenelzine ( Figures 5A and S3G) or the MAO-A irreversible selective inhibitor clorgyline ( Figures 5B and S3G), compared to control-treated cells. The impact of MAO inhibition on HCV replication was confirmed in Jc1-infected cells by quantifying viral RNA and protein levels ( Figure 5C,D), suggesting that MAO activity alleviates the inhibitory effect of catecholamines on HCV replication. These results, combined with data showing the DDC-mediated inhibition of HCV by L-Dopa and 5-HTP, suggest that the antiviral effect in hepatocytes is mediated at least by the accumulation of the endogenous produced and imported catecholamines.

Inhibition of MAO Monoamine Degradation Enzymes Reduces HCV Replication
Thus far, our data suggested that the accumulation of catecholamines in the cell cytosol negatively affected HCV replication. Part of the accumulated cytosolic-located catecholamines can either undergo auto-oxidation, producing toxic quinones that can also generate ROS [77], or can be deaminated by MAO enzymes. To examine if MAO function could contribute to the regulation of HCV replication, we performed an inhibition of MAO-A and MAO-B in Huh7.5 cells. MAO-A has a higher affinity to 5-HT, while both MAO isoforms metabolize catecholamines [20] and their inhibition causes the accumulation of these monoamines in the cytoplasm. We observed that virus replication was up to 2-fold lower in the presence of the MAO-A and MAO-B irreversible inhibitor phenelzine ( Figures 5A and S3G) or the MAO-A irreversible selective inhibitor clorgyline ( Figures 5B  and S3G), compared to control-treated cells. The impact of MAO inhibition on HCV replication was confirmed in Jc1-infected cells by quantifying viral RNA and protein levels

Induction of the Catecholamine and Serotonin Biosynthetic/Metabolic Pathway Down-Regulates HCV Replication
To further confirm the negative implication of the catecholamine producing enzymes on HCV, we applied inducers of protein kinase A (PKA) and protein kinase C (PKC). The activation of these proteins enhances the accumulation of catecholamines through phosphorylation of DDC [78], stimulation of TH transcription and enzymatic activity [12] or increasing MAO-B gene expression and activity [79]. Treatment of JcR2A-infected Huh7.5 cells for 72 h with different concentrations of PKA activator forskolin or PKC activator phorbol 12-myristate 13-acetate (PMA) resulted in up to a 2-fold reduction of virus-derived R-Luc activity ( Figures 6A,B and S3H). A PMA-dependent TH and MAO-B mRNA upregulation was observed in Huh7.5 cells (Figure 6C), confirming the effect of the protein kinase inducer on the catecholamine biosynthetic/metabolic pathway under our experimental conditions.
( Figure 5C,D), suggesting that MAO activity alleviates the inhibitory effect of catecholamines on HCV replication. These results, combined with data showing the DDC-mediated inhibition of HCV by L-Dopa and 5-HTP, suggest that the antiviral effect in hepatocytes is mediated at least by the accumulation of the endogenous produced and imported catecholamines.

Association of Catecholamine-Mediated HCV Regulation with Cellular Redox Homeostasis
Catecholamines in the cytoplasm are autoxidized, producing damaging quinones that also generate ROS. Auto-oxidation can be limited by MAO that oxidatively deaminate catecholamines and serotonin, generating H 2 O 2 [12,13,80]. Concerning HCV, there are reports showing an inhibitory effect of elevated ROS levels on HCV replication [81,82], as well as the sensitivity of viral core and NS5A proteins to oxidative stress-induced degradation [83]. cells for 72 h with different concentrations of PKA activator forskolin or PKC activator phorbol 12-myristate 13-acetate (PMA) resulted in up to a 2-fold reduction of virus-derived R-Luc activity ( Figures 6A,B and S3H). A PMA-dependent TH and MAO-B mRNA upregulation was observed in Huh7.5 cells (Figure 6C), confirming the effect of the protein kinase inducer on the catecholamine biosynthetic/metabolic pathway under our experimental conditions. Therefore, we sought to investigate whether the suppression of HCV replication by catecholamine biosynthesis is mediated by ROS production. First, we confirmed that the oxidative stress is induced in Huh7.5 cells treated with dopamine, norepinephrine or MAO inhibitors clorgyline and phenelzine, as detected by an increase in the gene expression of nuclear factor erythroid 2-related factor 2 (NRF2), which is a major cell survival factor under stress conditions, the NRF2-regulated antioxidant response gene heme oxygenase-1 (HO-1) [84], as well as the ROS-stimulated hypoxia inducible factor (HIF)-target VEGFA [85] ( Figure 7A-C). Treatment with catecholamines up-regulated HO-1 and VEGFA mRNA in Jc1-infected cells too. These findings are in accordance with previous data supporting that exogenously provided dopamine leads to oxidative stress responses evidenced by an increased expression of NRF2, HO-1 and hypoxia inducible factor-1α in human nonneuronal cells [86,87]. Viral infection also increased the expression of the antioxidant genes NRF2 and HO-1, as well as of VEGFA, as shown by comparing mock-treated infected to mock-infected cells ( Figure 7A,B), along with already published reports [88][89][90][91]. Then, as cells by utilizing reduced glutathione (GSH) diminish levels of H 2 O 2 and ROS [92], we evaluated a possible role of GSH in HCV replication under treatment with dopamine. Huh7.5 cells were inoculated with JcR2a or Jc1 for 4 h and then treated or not with DA in the presence or absence of GSH, using concentrations that do not impact cell growth ( Figure S3A,I), but influence oxidative stress-related gene expression ( Figure 7A,B,D). Glutathione alone enhanced HCV replication whereas, in combination with dopamine, it diminished the negative impact of dopamine on HCV replication ( Figure 7E,F) and in parallel, decreased the expression of oxidative stress-related genes ( Figure 7D). Accordingly, H 2 O 2 had a negative effect on Jc1 replication (i.e., negative and positive strand RNA amounts) ( Figure S6A), while it induced the antioxidant response ( Figure S6B). These results suggest that the cellular redox homeostasis mediates at least part of the role of the catecholamine biosynthesis and metabolism on HCV replication.

HCV Regulates the Expression of Catecholamine Biosynthesis Pathway-Related Enzymes
Based on the negative regulation of HCV replication exerted by the components of the catecholamine biosynthetic and metabolic pathway and on the effect of HCV on DDC expression, we investigated whether HCV, in turn, alters the expression of other enzymes of the pathway and examined further how HCV affects DDC protein. Concerning the latter, we aimed to elucidate the mechanism(s) mediating the down-regulation of DDC protein by HCV infection [7]. This includes the reduction of its total intracellular levels, detected in IF, as well as an accumulation of the~50 kDa DDC monomer with a concomitant reduction of a~120 kDa DDC immunoreactive SDS-resistant protein, which is possibly the dimeric catalytically active form of the protein or a yet unknown DDC isoform species [7,8]. As the subgenomic HCV JFH1 replicon, which expresses only the non-structural viral proteins, exerts the same effect on DDC as the full-length virus [7], we examined the role of individual non-structural proteins in modifying DDC levels. More specifically, we overexpressed NS4B and NS5A in Huh7-Lunet cells and analyzed their effects on DDC. These two proteins were selected as they are known to interact with multiple host proteins and modulate viral replication [93]. Western blot analysis showed that, similarly to the HCV virus and subgenomic replicon [7], NS4B caused an accumulation of the~50 kDa DDC monomer and a reduction of the~120 kDa DDC immunoreactive species ( Figure 8A). NS5A had the same phenotype only at the late time-point of 72 h post transfection, while it reduced the total DDC levels at earlier time-points ( Figure 8A). In agreement with the Western blot results, NS5A appeared to reduce DDC protein levels also in IF ( Figure 8B,C). Concerning its subcellular distribution, DDC did not colocalize significantly with NS5A (Pearson's correlation coefficient: R = 0.28 ± 0.10, Manders' colocalization coefficient: M1 = 0.23 ± 0.09), which is in consistence with the previously observed exclusion of DDC from the HCV replication sites [7] and (only partially) colocalized with NS4B (R = 0.37 ± 0.14, M1 = 0.59 ± 0.08) ( Figure 8D). However, both viral proteins failed to affect the transcription of DDC up to 72 h post-transfection ( Figure S4E). These results suggest that both viral proteins contribute to the phenotype of the virus-mediated negative regulation of DDC protein and that this phenotype is unrelated with the levels of expression of DDC.
Then, we investigated the impact of HCV infection on the expression levels of enzymes other than DDC in the monoamine biosynthetic and metabolic pathway. We observed that Jc1 infection in Huh7.5 cells suppressed both protein and mRNA levels of TH ( Figure 9A), which functions upstream of DDC by synthesizing L-Dopa. Similarly, a significant negative effect exerted by HCV was observed in both the protein and mRNA levels of DBH ( Figure  9B), which converts DA to NE. Based on our aforementioned finding that inhibition of DBH does not impact HCV ( Figure 4A-C), the negative effect of HCV on DBH might be indirect and result from the HCV-mediated down-regulation of the upstream biosynthetic enzyme DDC. Indeed, under conditions that DDC protein levels are reduced, as in the case of DDC silencing, a concomitant decrease of DBH levels occurs ( Figure S1E). Moreover, the levels of the monoamine transporter VMAT2 ( Figure 9C) were not altered during infection. In consistence with the tissue specificity of VMAT1 expression [18,94], its mRNA and protein were not detected in Huh7.5 cells ( Figure S7). Finally, viral infection did not affect monoamine oxidase MAO-A/B mRNA amounts ( Figure 9D,E), while it increased MAO-B at the protein level ( Figure 9E).  zymes other than DDC in the monoamine biosynthetic and metabolic pathway. We observed that Jc1 infection in Huh7.5 cells suppressed both protein and mRNA levels of TH ( Figure 9A), which functions upstream of DDC by synthesizing L-Dopa. Similarly, a significant negative effect exerted by HCV was observed in both the protein and mRNA levels of DBH ( Figure 9B), which converts DA to NE. Based on our aforementioned finding that inhibition of DBH does not impact HCV ( Figure 4A-C), the negative effect of HCV on DBH might be indirect and result from the HCV-mediated down-regulation of the upstream biosynthetic enzyme DDC. Indeed, under conditions that DDC protein levels are reduced, as in the case of DDC silencing, a concomitant decrease of DBH levels occurs ( Figure S1E). Moreover, the levels of the monoamine transporter VMAT2 ( Figure 9C) were not altered during infection. In consistence with the tissue specificity of VMAT1 expression [18,94], its mRNA and protein were not detected in Huh7.5 cells ( Figure S7). Finally, viral infection did not affect monoamine oxidase MAO-A/B mRNA amounts ( Figure  9D,E), while it increased MAO-B at the protein level ( Figure 9E). In total, these data highlight an interesting bidirectional relationship between HCV replication and catecholamine biosynthesis in hepatocytes, unraveling for the first time the role of this pathway in viral infections.

Discussion
In this study, we report for the first time that the biosynthesis and uptake of catecholamines restrict the replication of HCV in hepatocytes. This effect on HCV is strongly related with oxidative stress that is generated by the auto-oxidation of catecholamines in the cell cytosol. Indeed, all treatments that are expected to enhance the levels of catecholamines in the cell cytosol or inhibit catecholamine deamination induced an antioxidant cell response, and at the same time, down-regulated the replication of HCV. Vice versa, the use of antioxidants or treatments that lower the levels of catecholamines in the cell cytosol positively affected the replication of HCV. To counteract the effect of catecholamines, HCV has developed ways to reduce at least their synthesis in the cell. Actually, our results showed that HCV infection, apart from the already reported effects on the levels of DDC mRNA and protein, caused the down-regulation of TH that encodes the rate-limiting enzyme of catecholamine biosynthesis, and reduced the levels of DBH mRNA and protein, although chemical inhibition of DBH activity seemed not to affect the virus replication in hepatocytes. Moreover, viral replication increased the levels of the MAO-B enzyme that degrades catecholamines through deamination and limits their auto-oxidation. In parallel, our results showed that the effect of HCV on DDC protein can be partially reproduced by the sole expression of NS4B viral protein, while the expression of NS5A protein negatively affected the total levels of DDC similarly to viral infection.
Previous studies have reported the dysfunction of the dopamine and serotonin signaling pathways in the brain of HCV patients, correlating viral infection with neurological manifestations [95,96]. While the role of DDC expression regulation in the periphery remains largely unknown, novel research findings demonstrated the involvement of DDC in the apoptotic cell death of both neuronal and non-neuronal cells [10]. Previous studies from our group have also indicated the physical and functional association of DDC with PI3K, the kinase that phosphorylates AKT, regulating cell survival [11]. Moreover, our recent report was the first to associate HCV infection with the down-regulation of the catecholamine biosynthetic pathway in the liver, as data from HCV-infected cells and DDC overexpression or chemical inhibition studies in cultured hepatocytes had shown the existence of a bidirectional negative relationship between HCV replication and DDC mRNA protein expression, dependent at least on the association of DDC with PI3K [7,11].
In the present study, we confirmed the negative effect of DDC on HCV by performing DDC gene silencing in infectious and subgenomic replicon systems and verified that this effect is exerted at the level of viral RNA replication. Concerning the underlying mechanism of the observed influence of DDC on HCV, apart from the previously reported PI3K/AKT signaling-related mechanism [7,11], our results indicated that HCV is also suppressed by the intracellular processing of DDC substrates into products, as both L-Dopa and 5-HTP had a negative effect on HCV replication, which was significantly attenuated upon DDC silencing. Thus, in accordance with our findings from the treatment with the DDC inhibitor carbidopa [7], these data revealed the importance of the biosynthetic role of DDC on HCV replication. Interestingly, the application of L-Dopa positively affected the transcription of DDC ( Figure S4F) that putatively contributed as well to the effect on HCV. In addition to their biosynthesis, DDC products dopamine (DA) and serotonin (5-HT) also exerted a suppressive effect on HCV probably through their uptake, as their exogenous application in the cells was shown to reduce viral RNA and protein levels, while the use of the D2 dopamine receptor antagonist PCZ failed to reverse their effect on HCV. The latter suggested that the antiviral activity of the DDC biosynthetic pathway on HCV is not due to the autocrine/paracrine activation of the dopamine receptor D2, which has a well-characterized function in the liver, in inhibiting proliferation and migration and promoting apoptosis of HCC cells [63,64] and in regulating the detoxification function of hepatocytes [65]. The importance of the catecholamine uptake on HCV is also suggested by the results of treatment with the VMAT-specific, uptake and storage inhibitor reserpine [66][67][68][69][70][71][72][73]. Reserpine negatively affected the expression of OCT1, which encodes the major catecholamine transporter in hepatocytes [25,26], in parallel with the abrogation of the dopamine import in the cell. Accordingly, reserpine strongly induced the replication of HCV, while it abrogated the negative effect of the exogenously applied dopamine on the virus. Also, reserpine caused a down-regulation in the expression of DDC and DBH in infected cells, which is expected due to the higher levels of viral replication. Apart from the virus replication induction, a possible inhibition of the uptake of the cell culture medium containing L-Dopa, which is also a substrate of OCT1 [97], could account for the observed effect on these proteins. This is strengthened by the fact that the exogenously applied L-Dopa up-regulates DDC.
We further validated the negative relation of the catecholamine biosynthetic pathway with HCV infection by examining the implication of other proteins of the catecholamine biosynthesis (TH, DBH) and metabolism (MAO-A/B), applying their enzymatic products or using inhibitors of their activities.
Specifically, we focused first on norepinephrine (NE) and its biosynthetic enzyme DBH that synthesizes NE from DA inside the monoamine storage vesicles. Combined, our data, with the use of the DBH inhibitor nepicastat [75] and the catecholamine uptake and storage inhibitor reserpine, suggested that viral replication is suppressed by the uptake and presence of NE in the cell cytosol, however this cannot occur when NE is restricted inside the cytoplasmic vesicles. In contrast to DBH, which was shown to be expressed in Huh7.5 cells, in consistence with previous data from liver tissue [16], we did not detect, and thus did not study PNMT that converts norepinephrine to epinephrine, and this is in agreement with previous studies showing that the biosynthesis of epinephrine in the liver is accomplished by a non-specific methyltransferase and not PNMT [17].
The negative impact of catecholamines and serotonin on HCV was further supported by the use of the MAO inhibitors phenelzine and clorgyline, which suppress the oxidative deamination (degradation) of monoamines and thus increase their intracellular levels and autooxidation [12,13].
Our data showing that various steps of the biosynthetic and metabolic route of serotonin and catecholamines down-regulate HCV replication, were further supported by the negative effect of PKA and PKC inducers forskolin and PMA on the virus. These kinases up-regulate the biosynthetic enzymes DDC and TH [12,78] and the metabolic enzyme MAO [79].
Catecholamines that are not stored inside cytoplasmic vesicles or deaminated by MAO enzymes [12,13] can be auto-oxidated in the cell cytosol, producing toxic quinones that also generate ROS. This leads to oxidative stress, as has been observed by the induction of the expression of antioxidant genes (NRF2, HO-1) and the ROS stimulated HIF [86,87]. Our data confirmed that catecholamines induced the oxidative stress response gene expression (NRF2, HO-1 and VEGFA) in both infected and mock-infected Huh7.5 cells, as well as verified the negative role of MAO activity in this procedure. Interestingly, by the use of reduced GSH, we revealed that the down-regulation of HCV replication by catecholamines is strongly dependent by the catecholamine-related alterations on cell redox homeostasis. These results are in agreement with previous studies that suggest a negative effect of ROS on HCV replication [81][82][83]. Our results also indicated that the HCV infection itself also induced the expression of oxidative stress-related genes. This either could be attributed to a real oxidative stress generated by the virus replication [88][89][90][91] or could be part of a mechanism employed by the virus in order to shift the cell redox homeostasis to a more reduced environment aiming to favor viral replication. Evidence of the latter could be the results acquired by the sole use of GSH that positively affected the HCV replication.
Apart from the oxidative stress induced by catecholamines, which appears to control their antiviral effect, the implication of novel roles of these molecules remains to be elucidated. Recent studies showed that dopamine and serotonin covalently bind to histones regulating gene expression through a process called dopaminylation [98] and serotonylation [99], respectively. Moreover, the interaction of HCV with the monoamine biosynthetic and metabolic pathway may be related to the function of its products in cell proliferation and apoptosis of hepatocytes [28][29][30][31][32][33][34][35][36].
Furthermore, the ability of HCV to suppress the expression of the catecholamine biosynthetic enzymes DDC [7], TH and DBH, as well as to increase the levels of catecholamine degradation enzyme MAO-B, indicates that the virus counteracts this pathway for optimal replication. However, the virus did not alter the expression of the monoamine storage enzyme VMAT2, even though the inhibition of its activity impacted viral replication. Taking into account that TH is the rate-limiting enzyme of catecholamine biosynthesis [100], while DDC and DBH become rate-limiting under specific cases [78,101], it is possible that targeting the biosynthetic and metabolic enzymes is an efficient mechanism for the virus to resist the antiviral action of catecholamines.
Concerning DDC, we have previously shown that HCV mediates an accumulation of the~50 kDa DDC monomer with a concomitant reduction of a~120 kDa DDC complex, possibly corresponding to the dimeric catalytically active form of the protein [7,8]. Moreover, HCV was observed to reduce the total DDC intracellular levels in IF [7]. Towards the elucidation of the virus-mediated DDC down-regulation, here we showed that the non-structural viral proteins NS4B and NS5A are both implicated, but through different mechanisms. Specifically, NS4B simulates the effect of HCV on the accumulation of the 50 kDa DDC monomer and the decrease of the 120 kDa complex. However, in contrast to HCV infection, where DDC does not colocalize with the viral replication sites [7], DDC was partially colocalized with NS4B upon its overexpression. NS5A reduced total DDC protein levels and, late after transfection, when higher levels of the viral protein have been expressed, accumulated the 50 kDa monomer. Moreover, NS5A did not colocalize with DDC in the endoplasmic reticulum, in agreement with the infection assays [7]. However, although HCV reduces DDC mRNA [7], neither NS4B nor NS5A affected DDC mRNA levels, which suggests that they regulate DDC protein through post-transcriptional mechanisms. The colocalization of DDC subunits with NS4B may indicate an additional anti-viral mechanism which extends beyond that of dopamine biosynthesis. Based on this finding and on our earlier observation that viral infection disturbs DDC-PI3K complex formation [7,11], a possible NS4B-DDC interaction could hinder the collaboration of NS4B with PI3K (CLASS III VSP34) to facilitate viral replication [102]. In addition, NS5A may regulate DDC levels by promoting its degradation, as DDC has been implicated in programmed cell death [10] and NS5A is known to counteract apoptosis in infected cells by inducing the degradation of apoptosis-related proteins, such as p53 [103] and IP3R3 [104].
The research described in the manuscript has been limited in cell-culture based infectious models and thus an in vivo correlation between HCV infection and the expression of catecholamine biosynthetic pathway enzymes other than DDC remains to be studied. Moreover, any implication on HCV replication exerted by the function of catecholamines and serotonin in gene expression or in cell proliferation and apoptosis should be addressed.
The present study revealed an intricate bidirectional relationship between HCV and key molecules of the catecholamine and serotonin pathway, unraveling new determinants of viral replication and contributing to the increasing evidence for the role of bioactive amines in peripheral tissues.