Hepatitis C Virus Infection Induces the Beta Interferon Signaling Pathway in Immortalized Human Hepatocytes

ABSTRACT Beta interferon (IFN-β) expression is triggered by double-stranded RNA, a common intermediate in the replication of many viruses including hepatitis C virus (HCV). The recent development of cell culture-grown HCV allowed us to analyze the IFN signaling pathway following virus infection. In this study, we have examined the IFN-β signaling pathway following infection of immortalized human hepatocytes (IHH) with HCV genotype 1a (clone H77) or 2a (clone JFH1). We observed that IHH possesses a functional Toll-like receptor 3 pathway. HCV infection in IHH enhanced IFN-β and IFN-stimulated gene 56 (ISG56) promoter activities; however, poly(I-C)-induced IFN-β and ISG56 expression levels were modestly inhibited upon HCV infection. IHH infected with HCV (genotype 1a or 2a) exhibited various levels of translocation of IRF-3 into the nucleus. The upregulation of endogenous IFN-β and 2′,5′-oligoadenylate synthetase 1 mRNA expression was also observed in HCV-infected IHH. Subsequent studies suggested that HCV infection in IHH enhanced STAT1 and ISG56 protein expression. A functional antiviral response of HCV-infected IHH was observed by the growth-inhibitory role in vesicular stomatitis virus. Together, our results suggested that HCV infection in IHH induces the IFN signaling pathway, which corroborates observations from natural HCV infection in humans.

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Hepatitis C Virus Infection Impairs IRF-7 Translocation and Alpha Interferon Synthesis in Immortalized Human Hepatocytes

Journal of Virology ◽

10.1128/jvi.00900-10 ◽

2010 ◽

Vol 84 (21) ◽

pp. 10991-10998 ◽

Cited By ~ 31

Author(s):

Amit Raychoudhuri ◽

Shubham Shrivastava ◽

Robert Steele ◽

Srikanta Dash ◽

Tatsuo Kanda ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Signaling Pathway ◽

Nuclear Translocation ◽

Human Hepatocytes ◽

Hcv Infection ◽

Oligoadenylate Synthetase ◽

Downstream Signaling ◽

Inducible Protein ◽

Chronic Hcv

ABSTRACT Hepatitis C virus (HCV) establishes chronic infection in a significant number of infected humans, although the mechanisms for chronicity remain largely unknown. We have previously shown that HCV infection in immortalized human hepatocytes (IHH) induces beta interferon (IFN-β) expression (T. Kanda, R. Steele, R. Ray, and R. B. Ray, J. Virol. 81:12375-12381, 2007). However, the regulation of the downstream signaling pathway for IFN-α production by HCV is not clearly understood. In this study, the regulation of the IFN signaling pathway following HCV genotype 1a (clone H77) or genotype 2a (clone JFH1) infection of IHH was examined. HCV infection upregulated expression of total STAT1 but failed to induce phosphorylation and efficient nuclear translocation. Subsequent study revealed that HCV infection induces IFN-stimulated response element activation, as evidenced by upregulation of 2′,5′-oligoadenylate synthetase 1. However, nuclear translocation of IRF-7 was impaired following HCV infection. In HCV-infected IHH, IFN-α expression initially increased (up to 24 h) and then decreased at later time points, and IFN-α-inducible protein 27 was not induced. Interestingly, HCV infection blocked IRF-7 nuclear translocation upon poly(I-C) or IFN-α treatment of IHH. Together, our data suggest that HCV infection enhances STAT1 expression but impairs nuclear translocation of IRF-7 and its downstream molecules. These impairments in the IFN-α signaling pathway may, in part, be responsible for establishment of chronic HCV infection.

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Hepatitis C Virus Genotype 1a Growth and Induction of Autophagy

Journal of Virology ◽

10.1128/jvi.02093-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2241-2249 ◽

Cited By ~ 151

Author(s):

Malika Ait-Goughoulte ◽

Tatsuo Kanda ◽

Keith Meyer ◽

Jan S. Ryerse ◽

Ratna B. Ray ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Titer ◽

Infectious Hepatitis ◽

Autophagic Vacuole ◽

Hcv Infection ◽

Core Antigen ◽

Virus Genotype ◽

Hcv Genotype ◽

Genotype 1A

ABSTRACT We have previously reported that immortalized human hepatocytes (IHH) support the generation of infectious hepatitis C virus (HCV) genotype 1a (clone H77). In the present study, we have investigated the growth of HCV genotype 1a (clone H77) through serial passages and accompanying changes in IHH in response to infection. Eleven serial passages of HCV genotype 1a (clone H77) in IHH were completed. Virus replication was ascertained from the presence of HCV-specific sequences, the detection of core antigen, the virus genome copy number, and the virus titer in IHH culture fluid. Electron microscopy suggested that HCV infection induces autophagic vacuole formation in IHH. Fluorescence microscopy displayed localization of autophagic markers, microtubule-associated protein-1 light chain-3 and Apg5, on the vacuoles of HCV-infected hepatocytes. Taken together, our results suggested that HCV genotype 1a (clone H77) can be serially passaged in IHH and that HCV infection induces an autophagic response in hepatocytes.

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Hepatitis C Virus-Mediated Enhancement of MicroRNA miR-373 Impairs the JAK/STAT Signaling Pathway

Journal of Virology ◽

10.1128/jvi.03085-14 ◽

2015 ◽

Vol 89 (6) ◽

pp. 3356-3365 ◽

Cited By ~ 53

Author(s):

Anupam Mukherjee ◽

Adrian M. Di Bisceglie ◽

Ratna B. Ray

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Signaling Pathway ◽

Human Hepatocytes ◽

Hcv Infection ◽

Janus Kinase ◽

Hcv Rna ◽

Type I ◽

Primary Human Hepatocytes

ABSTRACTHepatitis C virus (HCV) is a serious global health problem and establishes chronic infection in a significant number of infected humans worldwide. Interferon (IFN) and IFN-stimulated genes (ISGs) are amplified during HCV infection but fail to eliminate virus from the liver in a large number of infected patients, and the mechanism is not fully understood. MicroRNAs (miRNAs) have been implicated in the control of many biological processes, including IFN signaling. To gain more insights into the role of cellular miRNAs in possible countermeasures of HCV for suppression of the host antiviral response, a miRNA array was performed by using primary human hepatocytes infected within vitrocell culture-grown HCV. A group of miRNAs were modulated in HCV-infected primary human hepatocytes. We focused on miR-373, as this miRNA was significantly upregulated in HCV-infected primary human hepatocytes. Here, we analyzed the function of miR-373 in the context of HCV infection. HCV infection upregulates miR-373 expression in hepatocytes and HCV-infected liver biopsy specimens. Furthermore, we discovered that miR-373 directly targets Janus kinase 1 (JAK1) and IFN-regulating factor 9 (IRF9), important factors in the IFN signaling pathway. The upregulation of miR-373 by HCV also inhibited STAT1 phosphorylation, which is involved in ISG factor 3 (ISGF3) complex formation and ISG expression. The knockdown of miR-373 in hepatocytes enhanced JAK1 and IRF9 expression and reduced HCV RNA replication. Taken together, our results demonstrated that miR-373 is upregulated during HCV infection and negatively regulated the type I IFN signaling pathway by suppressing JAK1 and IRF9. Our results offer a potential therapeutic approach for antiviral intervention.IMPORTANCEChronic HCV infection is one of the major causes of end-stage liver disease worldwide. Although the recent introduction of direct-acting antiviral (DAA) therapy is extremely encouraging, some infected individuals do not respond to this therapy. Furthermore, these drugs target HCV nonstructural proteins, and with selective pressure, the virus may develop a resistant strain. Therefore, understanding the impairment of IFN signals will help in designing additional therapeutic modalities. In this study, we provide evidence of HCV-mediated upregulation of miR-373 and show that miR-373 impairs IFN signaling by targeting JAK1/IRF9 molecules. The knockdown of miR-373 inhibited HCV replication by upregulating interferon-stimulating gene expression. Together, these results provided new mechanistic insights into the role of miR-373 in HCV infection and suggest a new potential target against HCV infection.

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Resistance Analysis of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00308-12 ◽

2012 ◽

Vol 56 (7) ◽

pp. 3670-3681 ◽

Cited By ~ 84

Author(s):

Fiona McPhee ◽

Jacques Friborg ◽

Steven Levine ◽

Chaoqun Chen ◽

Paul Falk ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protease Inhibitor ◽

Clinical Studies ◽

Replication Capacity ◽

Replication Complex ◽

Hcv Genotype ◽

Genotype 1A ◽

Genotype 1B ◽

Ns3 Protease

ABSTRACTAsunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) NS3 protease inhibitor demonstrating efficacy in alfa interferon-sparing, direct-acting antiviral dual-combination regimens (together with the NS5A replication complex inhibitor daclatasvir) in patients chronically infected with HCV genotype 1b. Here, we describe a comprehensivein vitrogenotypic and phenotypic analysis of asunaprevir-associated resistance against genotypes 1a and 1b using HCV replicons and patient samples obtained from clinical studies of short-term asunaprevir monotherapy. During genotype 1a resistance selection using HCV replicons, the primary NS3 protease substitutions identified were R155K, D168G, and I170T, which conferred low- to moderate-level asunaprevir resistance (5- to 21-fold) in transient-transfection susceptibility assays. For genotype 1b, a higher level of asunaprevir-associated resistance was observed at the same selection pressures, ranging from 170- to 400-fold relative to the wild-type control. The primary NS3 protease substitutions identified occurred predominantly at amino acid residue D168 (D168A/G/H/V/Y) and were associated with high-level asunaprevir resistance (16- to 280-fold) and impaired replication capacity. In asunaprevir single-ascending-dose and 3-day multiple-ascending-dose studies in HCV genotype 1a- or 1b-infected patients, the predominant pre-existing NS3 baseline polymorphism was NS3-Q80K. This substitution impacted initial virologic response rates in a single-ascending-dose study, but its effects after multiple doses were more ambiguous. Interestingly, for patient NS3 protease sequences containing Q80 and those containing K80, susceptibilities to asunaprevir were comparable when tested in an enzyme assay. No resistance-associated variants emerged in these clinical studies that significantly impacted susceptibility to asunaprevir. Importantly, asunaprevir-resistant replicons remained susceptible to an NS5A replication complex inhibitor, consistent with a role for asunaprevir in combination therapies.

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Hepatitis C Virus Induces Toll-Like Receptor 4 Expression, Leading to Enhanced Production of Beta Interferon and Interleukin-6

Journal of Virology ◽

10.1128/jvi.80.2.866-874.2006 ◽

2006 ◽

Vol 80 (2) ◽

pp. 866-874 ◽

Cited By ~ 131

Author(s):

Keigo Machida ◽

Kevin T. H. Cheng ◽

Vicky M.-H. Sung ◽

Alexandra M. Levine ◽

Steven Foung ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

B Cells ◽

Immune Responses ◽

Interleukin 6 ◽

Innate Immune ◽

Hcv Infection ◽

Innate Immune Responses ◽

Altered Expression ◽

Beta Interferon

ABSTRACT Hepatitis C virus (HCV) induces inflammatory signals, leading to hepatitis, hepatocellular carcinomas, and lymphomas. The mechanism of HCV involvement in the host's innate immune responses has not been well characterized. In this study, we analyzed expression and regulation of the entire panel of toll-like receptors (TLRs) in human B cells following HCV infection in vitro. Among all of the TLRs (TLRs 1 to 10) examined, only TLR4 showed an altered expression (a three- to sevenfold up-regulation) after HCV infection. Peripheral blood mononuclear cells from HCV-infected individuals also showed a higher expression level of TLR4 compared with those of healthy individuals. HCV infection significantly increased beta interferon (IFN-β) and interleukin-6 (IL-6) secretion from B cells, particularly after lipopolysaccharide stimulation. The increased IFN-β and IL-6 production was mediated by TLR4 induction, since the introduction of the small interfering RNA against TLR4 specifically inhibited the HCV-induced cytokine production. Among all of the viral proteins, only NS5A caused TLR4 induction in hepatocytes and B cells. NS5A specifically activated the promoter of the TLR4 gene in both hepatocytes and B cells. In conclusion, HCV infection directly induces TLR4 expression and thereby activates B cells, which may contribute to the host's innate immune responses.

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High prevalence of occult hepatitis C virus infection in patients with chronic hepatitis B virus infection

Journal of Medical Microbiology ◽

10.1099/jmm.0.058297-0 ◽

2013 ◽

Vol 62 (8) ◽

pp. 1235-1238 ◽

Cited By ~ 6

Author(s):

Inmaculada Castillo ◽

Javier Bartolomé ◽

Juan Antonio Quiroga ◽

Vicente Carreño

Keyword(s):

Chronic Hepatitis ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Hepatitis B Virus ◽

Virus Infection ◽

Hepatitis B ◽

Hcv Infection ◽

Hbv Infection ◽

Chronic Hepatitis B Virus ◽

B Virus

Hepatitis C virus (HCV) infection in the absence of detectable antibodies against HCV and of viral RNA in serum is called occult HCV infection. Its prevalence and clinical significance in chronic hepatitis B virus (HBV) infection is unknown. HCV RNA was tested for in the liver samples of 52 patients with chronic HBV infection and 21 (40 %) of them were positive for viral RNA (occult HCV infection). Liver fibrosis was found more frequently and the fibrosis score was significantly higher in patients with occult HCV than in negative ones, suggesting that occult HCV infection may have an impact on the clinical course of HBV infection.

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Screening for anti HCV

Annals of King Edward Medical University ◽

10.21649/akemu.v10i2.1179 ◽

2016 ◽

Vol 10 (2) ◽

Author(s):

Fatima Mehboob

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

Observational Study ◽

General Practitioners ◽

Clinical Features ◽

Screening Strategy ◽

Medical Ward ◽

Hcv Infection ◽

Hepatitis C Virus Infection

Purpose of this study is to evaluate the different indications for screening for Anti HCV. This study was carried out in outdoor and indoor department of North Medical Ward of Mayo Hospital, Lahore. This is a non-interventional observational study. Two hundred patients ELISA proved HCV infection were evaluated to find out what were the different circumstances or symptomatology when tests for HCV infection were advised. So that a screening strategy can be formed. As hepatitis C virus infection has varied presentation and clinical features, the general practitioners, physicians, dermatologists and psychiatrists should be conscious about it an advise for Anti HCV detection whenever it is suspected. Screening of the early cases is beneficial both for the patients and its relatives.

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In VitroActivity and Resistance Profile of Dasabuvir, a Nonnucleoside Hepatitis C Virus Polymerase Inhibitor

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04619-14 ◽

2014 ◽

Vol 59 (3) ◽

pp. 1505-1511 ◽

Cited By ~ 73

Author(s):

Warren Kati ◽

Gennadiy Koev ◽

Michelle Irvin ◽

Jill Beyer ◽

Yaya Liu ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Clinical Isolates ◽

Genotype 1 ◽

Subgenomic Replicon ◽

Hcv Genotype ◽

Genotype 1A ◽

Treatment Naïve ◽

Hcv Genotype 1 ◽

Hcv Ns5b

ABSTRACTDasabuvir (ABT-333) is a nonnucleoside inhibitor of the RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene. Dasabuvir inhibited recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates, with 50% inhibitory concentration (IC50) values between 2.2 and 10.7 nM, and was at least 7,000-fold selective for the inhibition of HCV genotype 1 polymerases over human/mammalian polymerases. In the HCV subgenomic replicon system, dasabuvir inhibited genotype 1a (strain H77) and 1b (strain Con1) replicons with 50% effective concentration (EC50) values of 7.7 and 1.8 nM, respectively, with a 13-fold decrease in inhibitory activity in the presence of 40% human plasma. This level of activity was retained against a panel of chimeric subgenomic replicons that contained HCV NS5B genes from 22 genotype 1 clinical isolates from treatment-naive patients, with EC50s ranging between 0.15 and 8.57 nM. Maintenance of replicon-containing cells in medium containing dasabuvir at concentrations 10-fold or 100-fold greater than the EC50resulted in selection of resistant replicon clones. Sequencing of the NS5B coding regions from these clones revealed the presence of variants, including C316Y, M414T, Y448C, Y448H, and S556G, that are consistent with binding to the palm I site of HCV polymerase. Consequently, dasabuvir retained full activity against replicons known to confer resistance to other polymerase inhibitors, including the S282T variant in the nucleoside binding site and the M423T, P495A, P495S, and V499A single variants in the thumb domain. The use of dasabuvir in combination with inhibitors targeting HCV NS3/NS4A protease (ABT-450 with ritonavir) and NS5A (ombitasvir) is in development for the treatment of HCV genotype 1 infections.

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Chronic Hepatitis C Virus Infection After Treatment for Pediatric Malignancy

Blood ◽

10.1182/blood.v90.3.1315 ◽

1997 ◽

Vol 90 (3) ◽

pp. 1315-1320 ◽

Cited By ~ 56

Author(s):

Simone Cesaro ◽

Maria Grazia Petris ◽

Flavio Rossetti ◽

Riccardo Cusinato ◽

Corrado Pipan ◽

...

Keyword(s):

Hepatitis C Virus ◽

Liver Disease ◽

Hepatitis C ◽

Virus Infection ◽

Chronic Liver Disease ◽

Chronic Liver ◽

Hcv Infection ◽

Pediatric Malignancy ◽

Infection Markers

Abstract Sera of 658 patients who had completed treatment for pediatric malignancy were analyzed by a second-generation enzyme-linked immunosorbent assay and recombinant immunoblot assay test to assess the prevalence of hepatitis C virus (HCV)-seropositivity. All HCV-seropositive patients underwent detailed clinical, laboratory, virologic, and histologic study to analyze the course of HCV infection. One hundred seventeen of the 658 patients (17.8%) were positive for HCV infection markers. Among the 117 anti-HCV+ patients, 41 (35%) were also positive for markers of hepatitis B virus infection with or without delta virus infection markers, 91 (77.8%) had previously received blood product transfusions, and 25 (21.4%) showed a normal alanine aminotransferase (ALT) level during the last 5-year follow-up (11 of them never had abnormal ALT levels). The remaining 92 patients showed ALT levels higher than the upper limit of normal range. Eighty-one of 117 (70%) anti-HCV+ patients were HCV-RNA+, with genotype 1b being present in most patients (54%). In univariate analysis, no risk factor for chronic liver disease was statistically significant. In this study, the prevalence of HCV infection was high in patients who were treated for a childhood malignancy. In about 20% of anti-HCV+ patients, routes other than blood transfusions are to be considered in the epidemiology of HCV infection. After a 14-year median follow-up, chronic liver disease of anti-HCV+ positive patients did not show progression to liver failure.

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