Hepatitis C Virus Replication In Hepatocytes Research Articles

CLEC18A Impairs Phagocytosis by Reducing FcγRIIA Expression and Arresting Autophagosome-Lysosome Fusion.

Mixed cryoglobulinemia (MC) is a hepatitis C virus (HCV)-related extrahepatic manifestation that is characterized by the abnormal presence of immune complexes (ICs). This may be due to the reduced uptake and clearance of ICs. The C-type lectin member 18A (CLEC18A) is a secretory protein that is expressed abundantly in hepatocytes. We previously observed that CLEC18A increased significantly in the phagocytes and sera of patients with HCV, particularly those with MC. Herein, we explored the biological functions of CLEC18A in the MC syndrome development of patients with HCV by using an in vitro cell-based assay with quantitative reverse transcription-PCR, immunoblotting, immunofluorescence, flow cytometry, and enzyme-linked immunosorbent assays. HCV infection or Toll-like receptor 3/7/8 activation could induce CLEC18A expression in Huh7.5 cells. Upregulated CLEC18A interacts with Rab5 and Rab7 and enhances type I/III interferon production to inhibit HCV replication in hepatocytes. However, overexpressed CLEC18A suppressed phagocytic activity in phagocytes. Significantly decreased levels of the Fc gamma receptor (FcγR) IIA were found in the neutrophils of HCV patients, particularly in those with MC (P < 0.005). We demonstrated that CLEC18A could inhibit FcγRIIA expression in a dose-dependent manner through the production of NOX-2-dependent reactive oxygen species to impair the uptake of ICs. Additionally, CLEC18A suppresses the Rab7 expression that is induced by starvation. Overexpressed CLEC18A does not affect autophagosome formation but does reduce the recruitment of Rab7 to autophagosomes, thereby retarding the maturation of autophagosomes and affecting autophagosome-lysosome fusion. We offer a novel molecular machinery with which to understand the association of HCV infection with autoimmunity and propose that CLEC18A may act as a candidate biomarker for HCV-associated MC. IMPORTANCE During infection, the host immune system produces cellular factors to protect against pathogen invasion. However, when the immune response overreacts and there is dysregulated cytokine homeostasis, autoimmunity occurs following an infection. We identified a cellular factor that is involved in HCV-related extrahepatic manifestation, namely, CLEC18A, which is expressed abundantly in hepatocytes and phagocytes. It inhibits HCV replication in hepatocytes by interacting with Rab5/7 and enhancing type I/III IFN expression. However, overexpressed CLEC18A inhibited FcγRIIA expression in phagocytes to impair phagocytosis. Furthermore, the interaction between CLEC18A and Rab5/7 may reduce the recruitment of Rab7 to autophagosomes and thereby retard autophagosome maturation and cause immune complex accumulation. A decreasing trend in CLEC18A levels that was accompanied by reduced HCV RNA titers and diminished cryoglobulin was observed in the sera of HCV-MC patients after direct-acting antiviral therapy. CLEC18A may be used for the evaluation of anti-HCV therapeutic drug effects and could be a potential predisposing factor for the development of MC syndrome.

HCV Infection Increases the Expression of ACE2 Receptor, Leading to Enhanced Entry of Both HCV and SARS-CoV-2 into Hepatocytes and a Coinfection State.

Recent studies suggest the enhancement of liver injury in COVID-19 patients infected with Hepatitis C virus (HCV). Hepatocytes express low levels of angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 entry receptor, raising the possibility of HCV-SARS-CoV-2 coinfection in the liver. This work aimed to explore whether HCV and SARS-CoV-2 coinfect hepatocytes and the interplay between these viruses. We demonstrate that SARS-CoV-2 coinfects HCV-infected Huh7.5 (Huh7.5HCV) cells. Both viruses replicated efficiently in the coinfected cells, with HCV replication enhanced in coinfected compared to HCV-mono-infected cells. Strikingly, Huh7.5HCV cells were eight fold more susceptible to SARS-CoV-2 pseudoviruses than naive Huh7.5 cells, suggesting enhanced SARS-CoV-2 entry into HCV-preinfected hepatocytes. In addition, we observed increased binding of spike receptor-binding domain (RBD) protein to Huh7.5HCV cells, as well as enhanced cell-to-cell fusion of Huh7.5HCV cells with spike-expressing Huh7.5 cells. We explored the mechanism of enhanced SARS-CoV-2 entry and identified an increased ACE2 mRNA and protein levels in Huh7.5HCV cells, primary hepatocytes, and in data from infected liver biopsies obtained from database. Importantly, higher expression of ACE2 increased HCV infection by enhancing its binding to the host cell, underscoring its role in the HCV life cycle as well. Transcriptome analysis revealed that shared host signaling pathways were induced in HCV-SARS-CoV-2 coinfection. This study revealed complex interactions between HCV and SARS-CoV-2 infections in hepatocytes, which may lead to the increased liver damage recently reported in HCV-positive COVID-19 patients. IMPORTANCE Here, we provide the first experimental evidence for the coexistence of SARS-CoV-2 infection with HCV, and the interplay between them. The study revealed a complex relationship of enhancement between the two viruses, where HCV infection increased the expression of the SARS-CoV-2 entry receptor ACE2, thus facilitating SARS-CoV-2 entry, and potentially, also HCV entry. Thereafter, SARS-CoV-2 infection enhanced HCV replication in hepatocytes. This study may explain the aggravation of liver damage that was recently reported in COVID-19 patients with HCV coinfection and suggests preinfection with HCV as a risk factor for severe COVID-19. Moreover, it highlights the possible importance of HCV treatment for coinfected patients. In a broader view, these findings emphasize the importance of identifying coinfecting pathogens that increase the risk of SARS-CoV-2 infection and that may accelerate COVID-19-related co-morbidities.

IP-10 Interferes With the Antiviral Response of Direct-Acting Antiviral Agents for Hepatitis C Virus Infection.

BackgroundIncreased interferon (IFN)-gamma inducible protein-10 (IP-10) level has been shown to be associated with sustained virologic responses (SVRs) to pegylated interferon-alpha 2a/ribavirin-based therapy in patients with chronic hepatitis C (CHC). We investigated the relationship between IP-10 and treatment response in patients with CHC treated with direct-acting antiviral agents (DAAs) therapy.MethodsWe measured the dynamic changes of IP-10 in samples from 90 patients with CHC. The serum IP-10 levels, intrahepatic expressions of IP-10 mRNA, and protein were determined, respectively. For the in vitro experiments, the expression changes of IP-10 in hepatitis C virus (HCV)-replicating Huh-7 cells with or without non-structural protein 5A (NS5A) inhibitor were analyzed using real-time reverse transcription-polymerase chain reaction and Western blotting.ResultsPatients with chronic hepatitis C had increased baseline IP-10 levels, intrahepatic IP-10 mRNA, and protein expression. After initiating DAAs therapy, serum IP-10 levels decreased gradually in patients who achieved cure, whereas in patients who failed the therapy, IP-10 levels did not change significantly or recovered from the initial decline. Multivariate logistic regression analysis confirmed that baseline IP-10 level ≤ 450 pg/ml and decline >30% at 12 weeks independently predicted the SVR in patients with CHC who received DAAs. In vitro, the expression of IP-10 mRNA and protein in HCV-replicating Huh-7 cells increased significantly. However, such activities were downregulated by NS5A inhibitor, followed by the reduction of HCV RNA levels and a decline in IP-10 levels.ConclusionIP-10 interfered with HCV replication in hepatocytes and the dynamic decline in IP-10 levels during DAA treatment predicted the SVR in patients with CHC.

IL-26 inhibits hepatitis C virus replication in hepatocytes

Interleukin-26 (IL-26) is a proinflammatory cytokine that has properties atypical for a cytokine, such as direct antibacterial activity and DNA-binding capacity. We previously observed an accumulation of IL-26 in fibrotic and inflammatory lesions in the livers of patients with chronic HCV infection and showed that infiltrating CD3+ lymphocytes were the principal source of IL-26. Surprisingly, IL-26 was also detected in the cytoplasm of hepatocytes from HCV-infected patients, even though these cells do not produce IL-26, even when infected with HCV. Based on this observation and possible interactions between IL-26 and nucleic acids, we investigated the possibility that IL-26 controlled HCV infection independently of the immune system. We evaluated the ability of IL-26 to interfere with HCV replication in hepatocytes and investigated the mechanisms by which IL-26 exerts its antiviral activity. We showed that IL-26 penetrated HCV-infected hepatocytes, where it interacted directly with HCV double-stranded RNA replication intermediates, thereby inhibiting viral replication. IL-26 interfered with viral RNA-dependent RNA polymerase activity, preventing the de novo synthesis of viral genomic single-stranded RNA. These findings reveal a new role for IL-26 in direct protection against HCV infection, independently of the immune system, and increase our understanding of the antiviral defense mechanisms controlling HCV infection. Future studies should evaluate the possible use of IL-26 for treating other chronic disorders caused by RNA viruses, for which few treatments are currently available, or emerging RNA viruses. This study sheds new light on the body's arsenal for controlling hepatitis C virus (HCV) infection and identifies interleukin-26 (IL-26) as an antiviral molecule capable of blocking HCV replication. IL-26, which has unique biochemical and structural characteristics, penetrates infected hepatocytes and interacts directly with viral RNA, thereby blocking viral replication. IL-26 is, therefore, a new player in antiviral defenses, operating independently of the immune system. It is of considerable potential interest for treating HCV infection and other chronic disorders caused by RNA viruses for which few treatments are currently available, and for combating emerging RNA viruses.

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.

Rituximab May Cause Increased Hepatitis C Virus Viremia in Rheumatoid Arthritis Patients Through Declining Exosomal MicroRNA-155.

Several studies have shown that rituximab may enhance hepatitis C virus (HCV) activity. MicroRNAs (miRNAs) have been implicated in modulating the host immune response in HCV infection; miRNAs can be packaged into the exosomes and then shuttled by the exosomes to aid biologic functions. However, the role of exosomal miRNAs (exo-miRNAs) in rituximab-related HCV activity enhancement remains unclear. The association between rituximab and increased HCV activity was examined using an in vitro cell-based assay. Purified exosomes were confirmed using immunoblotting and flow cytometry and quantified using enzyme-linked immunosorbent assay. Exosomal miRNA-155 (exo-miR-155) levels were measured using quantitative reverse transcription-polymerase chain reaction. In vitro data showed that B cell-derived miR-155 could inhibit HCV replication in hepatocytes through exosome transmission. Rituximab could both induce B cell depletion and affect intracellular miR-155 production as well as exo-miR-155 transmission and then enhance HCV activity in hepatocytes (P < 0.005). Serum exosome levels were increased in rheumatoid arthritis (RA) patients with HCV infection compared with the levels in RA patients without HCV infection (P < 0.01). The exo-miR-155 levels were significantly increased in RA patients with HCV infection compared with those without infection (P < 0.01). A significantly greater decrement of exo-miR-155 expression was observed after rituximab therapy compared with those observed before therapy (P < 0.01), and hepatitis C viral loads increased simultaneously (P < 0.05). Circulating exo-miR-155 levels were negatively correlated with hepatitis C viral loads and subsequently associated with rituximab-related HCV activity enhancement in RA patients. Exo-miR-155 may become a potential diagnostic biomarker or therapeutic target.

(-)-Epigallocatechin-3-gallate enhances poly I:C-induced interferon-λ1 production and inhibits hepatitis C virus replication in hepatocytes.

AIMTo investigate the effect of (-)-epigallocatechin-3-gallate (EGCG) on polyinosinic-polycytidylic acid (poly I:C)-triggered intracellular innate immunity against hepatitis C virus (HCV) in hepatocytes.METHODSA cell culture model of HCV infection was generated by infecting a hepatoma cell line, Huh7, with HCV JFH-1 strain (JFH-1-Huh7). Poly I:C with a high molecular weight and EGCG were used to stimulate the JFH-1-Huh7 cells. Real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of intracellular mRNAs and of intracellular and extracellular HCV RNA. Enzyme-linked immunosorbent assay was used to evaluate the interferon (IFN)-λ1 protein level in the cell culture supernatant. Immunostaining was used to examine HCV core protein expression in Huh7 cells.RESULTSOur recent study showed that HCV replication could impair poly I:C-triggered intracellular innate immune responses in hepatocytes. In the current study, we showed that EGCG treatment significantly increased the poly I:C-induced expression of Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and IFN-λ1 in JFH-1-Huh7 cells. In addition, supplementation with EGCG increased the poly I:C-mediated antiviral activity in JFH-1-Huh7 cells at the intracellular and extracellular HCV RNA and protein levels. Further investigation of the mechanisms showed that EGCG treatment significantly enhanced the poly I:C-induced expression of IFN-regulatory factor 9 and several antiviral IFN-stimulated genes, including ISG15, ISG56, myxovirus resistance A, and 2’-5’-oligoadenylate synthetase 1, which encode the key antiviral elements in the IFN signaling pathway.CONCLUSIONOur observations provide experimental evidence that EGCG has the ability to enhance poly I:C-induced intracellular antiviral innate immunity against HCV replication in hepatocytes.

Hepatitis C virus load in parenchyma cells correlates with hepatic injury in infected patients

The association between serum hepatitis C virus (HCV) load and hepatic injury in HCV-infected patients has been extensively investigated. The present study aimed to investigate the association between HCV load in hepatic parenchyma cells and hepatic injury in HCV-infected patients. A total of 56 HCV-infected patients were included in the present retrospective study. The serum HCV mRNA was determined using quantitative polymerase chain reaction, while the hepatic parenchyma cell volume and HCV mRNA in hepatic parenchyma cells were also determined. Hepatic injury was evaluated on the basis of the severity of inflammation and fibrosis. The results demonstrated that there were evident differences in the mean serum HCV RNA levels and the HCV load/parenchyma cell volume among the various grades of hepatic inflammation (G1-G4) when groups with the least and most inflammation were compared (G1 vs. G4; P<0.05). Significant differences in the HCV load existed between groups divided according to the fibrosis grade; in addition, differences existed between fibrosis grades S1 and S2, and S2 and S4 when comparing serum HCV RNA levels (P<0.05). Similarly, differences existed between every two fibrosis stages (S0 vs. S4, S2 vs. S3, and S2 vs. S4; P<0.05) when viral loads and parenchyma cell volumes were compared (F=2.860, P<0.05). Furthermore, the fibrosis staging was correlated with the viral load/parenchyma cell volume (F=2.670, P<0.05). In conclusion, hepatic fibrosis grade was found to be associated with HCV load in parenchyma cells. The results of the present study demonstrated that the viral load in parenchyma cells is a more appropriate index compared with the serum viral load for evaluating HCV replication in hepatocytes, and may function as an important factor in HCV-infected hepatic injury evaluation.

HIV Vpr protein upregulates microRNA-122 expression and stimulates hepatitis C virus replication.

Human immunodeficiency virus (HIV)/hepatitis C virus (HCV) co-infection is characterized by higher serum HCV RNA loads compared with HCV mono-infection. However, the relationship between HIV and HCV replication remains to be clarified. HIV Vpr has been shown to play an essential role in HIV replication. In this study, we aimed to explore the role of Vpr in HCV replication and pathogenesis. We therefore used the genotype 2a full-length HCV strain JFH1 infection system and the genotype 1b full-length HCV replicon OR6 cell line to analyse the effects of Vpr on HCV replication. We found that Vpr promoted HCV 5′ UTR activity, HCV RNA replication and HCV protein expression in two HCV infection cell models. Additionally, lymphocyte-produced Vpr significantly induced HCV 5′ UTR activity and HCV replication in hepatocytes. We also found that Vpr upregulated the expression of miR-122 by stimulating its promoter activity. Furthermore, an miR-122 inhibitor suppressed the Vpr-mediated enhancement of both HCV 5′ UTR activity and HCV replication. In summary, our results revealed that the Vpr-upregulated expression of miR-122 is closely related to the stimulation of HCV 5′ UTR activity and HCV replication by Vpr, providing new evidence for how HIV interacts with HCV during HIV/HCV co-infection.

Antiviral and Immunoregulatory Effects of Indoleamine-2,3-Dioxygenase in Hepatitis C Virus Infection

In patients with hepatitis C virus (HCV) infection, enhanced activity of indoleamine-2,3-dioxygenase 1 (IDO) has been reported. IDO - a tryptophan-catabolizing enzyme - has been considered as both an innate defence mechanism and an important regulator of the immune response. The molecular mechanism of IDO induction in HCV infection and its role in the antiviral immune response remain unknown. Using primary human hepatocytes, we show that HCV infection stimulates IDO expression. IDO gene induction was transient and coincided with the expression of types I and III interferons (IFNs) and IFN-stimulated genes in HCV-infected hepatocytes. Overexpression of hepatic IDO prior to HCV infection markedly impaired HCV replication in hepatocytes, suggesting that IDO limits the spread of HCV within the liver. siRNA-mediated IDO knock-down revealed that IDO functions as an IFN-mediated anti-HCV effector. Hepatic IDO was most potently induced by IFN-γ, and ongoing HCV replication could significantly upregulate IDO expression. IRF1 (IFN-regulatory factor 1) and STAT1 (signal transducer and activator of transcription 1) regulated hepatic IDO expression. Hepatic IDO expression also had a significant inhibitory effect on CD4+ T-cell proliferation. Our data suggest that hepatic IDO plays a dual role during HCV infection by slowing down viral replication and also regulating host immune responses.

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Evaluation of Effect of Low-Density Lipoprotein Levels on the Measurement of Hepatitis C Viral Load in Chronic Hepatitis C Patients

Background: Low density lipoprotein receptor (LDLR) has been proposed as a candidate receptor for hepatitis C virus (HCV). Free beta-lipoproteins in a human serum may regulate the rate of hepatocyte infection by competing with the virus. Therefore, serum HCV levels should be regulated by rise and fall of serum beta-lipoproteins since the infection rate of virions influences HCV replication in hepatocytes and release of virions by hepatocytes. Aim: To evaluate effect of low-density lipoprotein levels on the measurement of hepatitis C viral load in chronic hepatitis C patients and estimate the levels of viral load of hepatitis C in relation to variation of the corresponding low-density lipoprotein levels in chronic hepatitis C patients and their relations to each other. Subjects and Methods: 30 HCV hyperlipidemic patients were subjected to clinical evaluation and laboratory investigations included follow up by repeated measurements of LDL level, AST, ALT, and Albumin for patient selection. Results: There was an inverse statistically significant correlation (r = -0.388, p=0.034) between the HCV RNA absolute viral load differences and LDL level absolute differences (the lower the LDL differences, the higher the HCV RNA differences). Also, there was an inverse statistically significant correlation (r=-0.42, p=0.021) between HCV RNA absolute differences and LDL level percent differences (the higher the LDL percent differences, the lower the HCV RNA absolute differences).The predictive variables for HCV RNA viral load level differences were LDL level differences and LDL percent differences whereas AST, ALT, and Albumin differences were not. Conclusion: Low-density lipoprotein levels have an effect on the measurement of hepatitis C viral load in chronic hepatitis C patients. Therefore, this must be taken in consideration when chronic hepatitis C patients have a history of dyslipidemia and perform a HCV RNA viral load by PCR.

Hepatic stellate cells, liver innate immunity, and hepatitis C virus

Chronic hepatitis C virus (HCV) infection can cause liver damage, ranging from mild to more severe conditions, such as fibrosis and cirrhosis. Hepatic stellate cell (HSC) activation is a key event in HCV-induced liver fibrosis. HSCs express several HCV coreceptors that interact with HCV proteins, promoting liver fibrogenesis. In addition, HSCs have the ability to engulf apoptotic bodies of hepatocytes induced by HCV and trigger a profibrogenic response. Recent studies have suggested that HSCs may play a novel role in the liver innate immunity. HSCs enhanced differentiation and accumulation of regulatory T cells. HSCs-activated natural killer cells could produce γ-interferon that inhibits HCV replication. Importantly, HSCs possess functional Toll-like receptor-3 and retinoic acid-inducible gene I that can be activated by their ligands (poly I :C, 5'ppp-dsRNA), leading to the induction of interferon and inhibition of HCV replication in hepatocytes. These new observations highlight the importance of HSCs in liver immunity against HCV, which is the focus of this review paper.

CD40 inhibits replication of hepatitis C virus in primary human hepatocytes by c-jun N terminal kinase activation independent from the interferon pathway

CD40, a member of the tumor necrosis factor receptor family, and its ligand, CD40L (CD154), are important regulators of the antiviral immune response. CD40L is up-regulated on lymphocytes and CD40 on hepatocytes during infection with hepatitis C virus (HCV); we investigated the role of CD40 signaling during HCV replication in hepatocytes. Viral replication was studied in primary human hepatocytes (PHH) and Huh7.5 cells using the infectious HCV Japanese fulminate hepatitis 1 isolate (JFH1) culture system, and in coculture with HCV antigen-specific CD8+ T cells. CD40L rapidly and transiently inhibits expression of the HCV nonstructural proteins NS3 and NS5A as well as HCV structural proteins core and E2 in Huh7.5 cells. Similarly, CD40L prevented replication of HCV in PHH, in synergy with interferon (IFN)-alpha. In Huh7.5 cells with replicating HCV, CD40L prevented production of infectious viral particles. When HCV antigen-specific CD8+ T cells were cocultured with HLA-A2-expressing Huh7 cells that had replicating virus, the T cells became activated, up-regulated CD40L, and inhibited HCV replication. Inhibition of CD40L partially prevented the antiviral activity of the CD8+ T cells. The antiviral effect of CD40L required activation of c-Jun N terminal kinases (JNK)1/2, but not induction of apoptosis or the JAK/STAT pathway that is necessary for the antiviral effects of IFNs. CD40 inhibits HCV replication by a novel, innate immune mechanism. This pathway might mediate viral clearance, and disruptions might be involved in the pathogenesis of HCV infection.

Retinoic acid inducible gene-I (RIG-I) signaling of hepatic stellate cells inhibits hepatitis C virus replication in hepatocytes

Retinoic acid inducible gene-I (RIG-I) is critical in the activation of the type I IFN-dependent antiviral innate immune response to hepatitis C virus (HCV) infection. We examined whether hepatic stellate cells (HSC; LX-2) possess a functional RIG-I signaling pathway and produce antiviral factors that can inhibit HCV. We showed that LX-2 cells treated with the RIG-I ligand (5'ppp-dsRNA) expressed significantly higher levels of IFN-β and IFN-λ than the control cells. The RIG-I activation in LX-2 cells also induced the expression of Toll-like receptor 3 (TLR3) and IFN regulatory factor-7 (IRF-7), the key regulators of the IFN signaling pathway. When HCV Japanese fulminant hepatitis (JFH)-1-infected hepatocytes were co-cultured with LX-2 cells stimulated with 5'ppp-dsRNA or incubated in media conditioned with supernatant (SN) from 5'ppp-dsRNA-stimulated LX-2 cells, HCV replication in hepatocytes was suppressed significantly. This LX-2 cell action on HCV replication was mediated through both IFN-β and IFN-λ, as Abs to IFN-α/β or IFN-λ receptors could neutralize the LX-2 SN-mediated anti-HCV effect. The role of IFNs in LX-2 cell-mediated anti-HCV activity is further supported by the observation that LX-2 SN treatment induced the expression of IFN stimulated genes, 2'-5'-oligoadenylate synthase-1 (OAS-1) and myxovirus resistance A (MxA), in HCV-infected Huh7 cells. These observations highlight the importance of HSC in liver innate immunity against HCV infection via a RIG-I-mediated signaling pathway.

Ribavirin Enhances the Action of Interferon-α against Hepatitis C Virus by Promoting the p53 Activity through the ERK1/2 Pathway

Background/AimsRibavirin significantly enhances the antiviral response of interferon-α (IFN-α) against Hepatitis C virus (HCV), but the underlying mechanisms remain poorly understood. Recently, p53 has been identified as an important factor involving the suppression of HCV replication in hepatocytes. We, therefore, decided to investigate whether and how ribavirin inhibits the replication of HCV by promoting the activity of p53.MethodsHepG2 and HCV replicons (JFH1/HepG2) were utilized to study the relationship between ribavirin and p53. The effect of ribavirin on cell cycles was analyzed by flow cytometry. The activation of p53 and the signaling pathways were determined using immunoblotting. By knocking down ERK1/ERK2 and p53 utilizing RNA interference strategy, we further assessed the role of ERK1/2 and p53 in the suppression of HCV replication by ribavirin in a HCV replicon system.ResultsUsing HepG2 and HCV replicons, we demonstrated that ribavirin caused the cell cycle arrest at G1 phase and stabilized and activated p53, which was associated with the antiviral activity of ribavirin. Compared to either ribavirin or IFN-α alone, ribavirin plus IFN-α resulted in greater p53 activation and HCV suppression. We further identified ERK1/2 that linked ribavirin signals to p53 activation. More importantly, knockdown of ERK1/2 and p53 partially mitigated the inhibitory effects of ribavirin on the HCV replication, indicating that ERK1/2-p53 pathway was involved in the anti-HCV effects of ribavirin.ConclusionRibavirin stimulates ERK1/2 and subsequently promotes p53 activity which at least partly contributes to the enhanced antiviral response of IFN-α plus ribavirin against HCV.

RNA-Sequencing Analysis of 5' Capped RNAs Identifies Many New Differentially Expressed Genes in Acute Hepatitis C Virus Infection

We describe the first report of RNA sequencing of 5' capped (Pol II) RNAs isolated from acutely hepatitis C virus (HCV) infected Huh 7.5 cells that provides a general approach to identifying differentially expressed annotated and unannotated genes that participate in viral-host interactions. We identified 100, 684, and 1,844 significantly differentially expressed annotated genes in acutely infected proliferative Huh 7.5 cells at 6, 48, and 72 hours, respectively (fold change ≥ 1.5 and Bonferroni adjusted p-values < 0.05). Most of the differentially expressed genes (>80%) and biological pathways (such as adipocytokine, Notch, Hedgehog and NOD-like receptor signaling) were not identified by previous gene array studies. These genes are critical components of host immune, inflammatory and oncogenic pathways and provide new information regarding changes that may benefit the virus or mediate HCV induced pathology. RNAi knockdown studies of newly identified highly upregulated FUT1 and KLHDC7B genes provide evidence that their gene products regulate and facilitate HCV replication in hepatocytes. Our approach also identified novel Pol II unannotated transcripts that were upregulated. Results further identify new pathways that regulate HCV replication in hepatocytes and suggest that our approach will have general applications in studying viral-host interactions in model systems and clinical biospecimens.

Morphine Inhibits Intrahepatic Interferon‐α Expression and Enhances Complete Hepatitis C Virus Replication

Heroin addicts are a high-risk group for hepatitis C virus (HCV) infection and the development of chronic HCV disease. We thus examined whether morphine, the active metabolite of heroin, has the ability to inhibit intrahepatic interferon (IFN)- alpha expression, facilitating HCV replication in human hepatocytes. Morphine inhibited intrahepatic IFN- alpha expression, which was associated with an increase in HCV replication in hepatocytes. Moreover, morphine compromised the anti-HCV effect of recombinant IFN- alpha . Investigation of the mechanism responsible for the morphine action revealed that morphine inhibited expression of IFN regulatory factor 5 in the hepatocytes. In addition, morphine suppressed the expression of p38, an important signal-transducing molecule involved in IFN- alpha -mediated anti-HCV activity. These findings indicate that morphine plays a cofactor role in facilitating HCV persistence in human hepatocytes.

Adenovirus-mediated gene transfer of interferon α inhibits hepatitis C virus replication in hepatocytes

Recently we reported that on-site interferon (IFN)-α production in the liver using an adenovirus vector can achieve a substantial confinement of IFN-α in the target organ and can improve liver fibrosis in a rat liver cirrhosis model. However, the major therapeutic effect of IFN for hepatitis C virus (HCV)-associated liver diseases is its antiviral effect on HCV. As a prelude to the in vivo HCV infection experiment using a primate animal model, here we examined the antiviral effect of IFN-α gene transfer into HCV-positive hepatocytes in vitro. The non-neoplastic human hepatocyte cell line PH5CH8 was inoculated with HCV-positive serum. Successful in vitro HCV replication and thus the validity of this model was confirmed by a strong selection for HCV variants determined by sequence analysis of the hypervariable region and an increase of HCV RNA estimated by real time TaqMan RT-PCR. One day after the inoculation of HCV, PH5CH8 cells were infected with adenoviral vectors encoding human IFN-α cDNA. HCV completely disappeared 9 days after the adenoviral infection, which is linked to the increase of 2 ′,5 ′-oligoadenylate synthetase activity, suggesting that IFN-α produced by gene transfer effectively inhibits HCV replication in hepatocytes. This study supports the development of IFN-α gene therapy for HCV-associated liver diseases.