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Background: In patients with hepatitis C virus (HCV) and malignant lymphoma, hepatitis C flare during R-CHOP can result in discontinuation of treatment. However, appropriate therapeutic strategies for managing hepatitis C flare during R-CHOP have not been established, and this issue is complicated by conflicting results regarding the use of direct-acting antivirals in patients with uncontrolled malignancies., Case Presentation: We report the first case of effective and safe treatment with on-demand 8-week glecaprevir and pibrentasvir for hepatitis C flare during R-CHOP in a patient with diffuse large B-cell lymphoma (DLBCL). The patient completed five additional courses of R-CHOP without hepatic toxicity. A complete response of DLBCL and a sustained virological response were observed at 24 weeks after glecaprevir and pibrentasvir completion., Conclusion: On-demand, direct-acting antivirals could be a novel strategy for managing hepatitis C flare during R-CHOP.

Background: The efficacy, safety, and pharmacokinetics of the combination of three direct-acting antiviral (DAA) agents (adafosbuvir [also known as AL-335], odalasvir, and simeprevir) were investigated in DAA treatment-naïve Japanese patients with genotype (GT)1 or GT2 chronic hepatitis C virus (HCV) infection, with or without compensated cirrhosis., Methods: In this Phase IIa, open-label, multicenter study-OMEGA-3 (NCT02993250)-patients received JNJ-4178 (adafosbuvir 800 mg once daily [QD], odalasvir 25 mg QD, and simeprevir 75 mg QD) for 8 (non-cirrhotic patients; Cohort 1) or 12 (cirrhotic patients; Cohort 2) weeks. Patients were followed-up to 24 weeks following the end of treatment (EOT). The primary endpoint was safety, including adverse events (AEs)., Results: Overall, 33 patients were enrolled into Cohort 1 (N = 22) or 2 (N = 11) and received combined treatment with JNJ-4178. During the treatment and follow-up phases, a higher percentage of patients in Cohort 2 (81.8%) experienced AEs compared with Cohort 1 (68.2%), but the incidence of treatment-related AEs was similar. Most AEs were mild-to-moderate in severity and no patients discontinued due to an AE. There was one serious AE (cataract) in a patient in Cohort 2, which was not considered related to treatment. All patients achieved sustained virologic response 12 weeks after EOT (SVR12). No incidences of viral relapse were observed during follow-up., Conclusions: In HCV GT1- and GT2-infected Japanese patients, treatment with JNJ-4178 was well tolerated and resulted in 100% of patients achieving SVR12.

Background: MicroRNAs (miRNAs) have gained much attention as cellular factors regulating hepatitis C virus (HCV) infection. miR-27b has been shown to regulate HCV infection in the hepatocytes via various mechanisms that have not been fully elucidated. In this study, therefore, we examined the mechanisms of miR-27b-mediated regulation of HCV infection., Methods: In silico screening analysis, transfection with miR-27b mimic, and a cell-based reporter assay were performed to identify miR-27b target genes. Cell cultured-derived HCV (HCVcc) was added to Huh7.5.1 cells knocked down for aquaporin (AQP) 11 (AQP11) and overexpressing AQP11. HCV replication levels were evaluated by real-time RT-PCR analysis of HCVcc genome., Results: Infection of Huh7.5.1 cells with HCVcc resulted in significant elevation in miR-27b expression levels. In silico analysis revealed that AQP11, which is an AQP family member and is mainly localized in the endoplasmic reticulum (ER), was a candidate for a target gene of miR-27b. Transfection of a miR-27b mimic significantly reduced AQP11 expression, but a cell-based reporter assay demonstrated that miR-27b did not suppress the expression of a reporter gene containing the 3'-untranslated region of the AQP11 gene, suggesting that miR-27b indirectly suppressed AQP11 expression. AQP11 expression levels were significantly reduced by infection with HCVcc in Huh7.5.1 cells. Knockdown and over-expression of AQP11 significantly reduced and increased HCVcc genome levels in the cells following infection, respectively, however, AQP11 knockdown did not show significant effects on the HCVcc titers in the culture supernatants., Conclusions: These results indicated that HCV infection induced a miR-27b-mediated reduction in AQP11 expression, leading to a modest reduction in HCV genome levels in the cells, not HCV titers in the culture supernatants.

Clinical trials and real-world data have proven that hepatitis C virus (HCV) in most infected patients can be eradicated by direct-acting antivirals (DAAs). However, the proper retreatment regimen for hemodialysis patients with HCV infection who have previously failed to respond to DAAs has not been clarified. We herein report, for the first time, the successful retreatment with glecaprevir and pibrentasvir, of three hemodialysis patients with genotype 1 or 2 HCV infection, who had previously failed to respond to combination therapy with an HCV-NA5A inhibitor (daclatasvir) and an HCV protease inhibitor (asunaprevir).

Although interferon (IFN)‑based treatment of patients with chronic hepatitis C virus (HCV) infection is widely applied, treatment resistance is often observed in patients with advanced liver fibrosis. Given that the molecular mechanisms of IFN resistance in liver fibrosis remain elusive, the present study investigated the effects of extracellular matrix (ECM) on IFN signaling in hepatic cells. The native HuH‑7 human hepatoma cell line and HuH‑7 cells were stably transfected with full‑length HCV‑RNA fused with Renilla luciferase (OR6 cells) were cultured on ECM‑coated dishes or non‑coated plastic dishes (NDs), and treated with human IFN‑α. In Huh‑7 cells cultured on coated dishes, the IFN‑stimulated response element (ISRE) luciferase activity was measured following ISRE plasmid transfection and the expression of IFN‑stimulated genes (ISG) were significantly lower than those in cells cultured on NDs. In addition, after IFN‑α treatment, the amount of HCV‑RNA and viral protein produced by OR6 cells cultured on coated dishes was higher than that produced by cells cultured on NDs. When cells were treated with β1‑integrin‑blocking antibody to disrupt the cell‑matrix interaction, the ISRE luciferase activity was restored, and the protein expression of ISG was increased, while that of HCV proteins was suppressed. Treatment of cells with integrin‑linked kinase (ILK) inhibitor or focal adhesion kinase (FAK) inhibitor restored the ISRE luciferase activity and expression of ISG proteins. These results suggested that β1‑integrin‑mediated signals affected the IFN signaling and promoted HCV replication. Therefore, the accumulation of ECM in liver fibrosis may impair IFN signaling through β1‑integrin‑mediated signaling involving ILK and FAK.

Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. In this study, we aimed to further characterize the role of macrophage-derived EVs in immune responses against hepatitis C virus (HCV) and the potential of polyunsaturated fatty acids (PUFAs) to modulate this modality of innate immunity. To this end, EVs were isolated from interferon-stimulated macrophage cultures or from serum of patients with acute or chronic hepatitis C. EVs were characterized by electron microscopy, flow cytometry, RNA-sequencing, and Western blot analysis. The effect of EVs on replication of HCV was assessed in coculture models. Functional analyses were performed to assess the impact of PUFAs on EV-mediated antiviral immunity. We found that macrophages secreted various cytokines shortly after stimulation with type I and II IFN, which orchestrated a fast but short-lasting antiviral state. This rapid innate immune answer was followed by the production of macrophage-derived EVs, which induced a late, but long-lasting inhibitory effect on HCV replication. Of note, exposure of macrophages to PUFAs, which are important regulators of immune responses, dampened EV-mediated antiviral immune responses. Finally, EVs from patients with hepatitis C exhibited long-lasting antiviral activities during IFN therapy as well. The antiviral effect of EVs from Caucasian and Japanese patients differed, which may be explained by different nutritional uptake of PUFAs. In conclusion, our data indicate that macrophage-derived EVs mediate long-lasting inhibitory effects on HCV replication, which may bridge the time until efficient adaptive immune responses are established, and which can be blunted by PUFAs.

Hepatitis C virus (HCV) infection is a major cause of liver-related morbidity and mortality. In order to develop effective remedies for hepatitis C, it is important to understand the HCV infection profile and host-HCV interaction. HCV-induced innate immune responses play a crucial role in spontaneous HCV clearance; however, HCV-induced innate immune responses have not been fully evaluated in hepatocytes, partly because there are few in vitro models of HCV-induced innate immunity. Recently, human induced pluripotent stem (iPS) cells have received much attention as an in vitro model of infection with various pathogens, including HCV. We previously established highly functional hepatocyte-like cells differentiated from human iPS cells (iPS-HLCs). Here, we examined the potential of iPS-HLCs as an in vitro HCV infection model, especially for evaluation of the relationship between HCV infection levels and HCV-induced innate immunity. Significant expressions of type I and III interferons (IFNs) and IFN-stimulated genes (ISGs) were induced following transfection with HCV genomic replicon RNA in iPS-HLCs. Following inoculation with the HCV JFH-1 strain in iPS-HLCs, peaks of HCV genome replication and HCV protein expression were observed on day 2, and then both the HCV genome and protein levels gradually declined, while the mRNA levels of type III IFNs and ISGs peaked at day 2 following inoculation. These results suggest that the HCV genome efficiently replicates in iPS-HLCs, resulting in HCV genome-induced up-regulation of IFNs and ISGs, and thereafter, HCV genome-induced up-regulation of IFNs and ISGs mediates a reduction in the HCV genome and protein levels in iPS-HLCs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus with an estimated infection in ∼180 million people worldwide, and its chronic infection leads to development of cirrhosis and hepatocellular carcinoma. Although recent development of direct acting antiviral (DAA) compounds improved anti-HCV regimens, alternative therapeutic compounds are still demanded due to an expected emergence of escape mutants for those DAAs. In order to identify novel anti-HCV agents, we conducted chemical library screening for 2086 compounds using HCV Rep-Feo reporter replicon in Huh7 hepatoma cells. Our screening identified retinoid derivative Tp80, which inhibits replication of HCV Rep-Feo (genotype 1b) and JFH1 HCV (genotype 2a) with 0.62 μM and 1.0 μM, respectively, of 50% effective concentration (EC 50 ), at which cytotoxicity is not evident for host hepatocytes. Subsequent transcriptome profiling revealed Tp80 exhibits anti-HCV activity through restoration of gastrointestinal glutathione peroxidase (GI-GPx), suppression of which is responsible for HCV-induced oxidative stress to facilitate HCV replication. Furthermore, comparison of Tp80 with other retinoid derivatives revealed Tp80 shows best potency in both GI-GPx restoration and anti-HCV activity among compounds we examined. In conclusion, our current study provides Tp80 as a promising candidate of anti-HCV compound, suppressing host cellular oxidative stress through a restoration of GI-GPx., (© 2017 Wiley Periodicals, Inc.)

Hepatitis C virus (HCV) infection remains the main cause of liver disease and can lead to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV may also develop extrahepatic manifestations in the skin, eyes, joints, kidneys, nervous system, and immune system. In fact, several studies reported that up to 70% of HCV patients experienced extrahepatic manifestations. Lichen planus (LP), which is an immune system disorder that is triggered by viral infections, allergens, and stress, can affect the skin, mouth, nails, and scalp. The association of LP with HCV has been reported, but the effect of HCV treatment on LP remission is controversial. We encountered a 53-year-old man with HCV genotype 2a and LP that were successfully treated with sofosbuvir and ribavirin for 12 weeks. After treatment, he achieved sustained virological response against HCV and remission of erosive LP lesions on the lip. In the era of interferon (IFN)-based treatment for HCV, exacerbation of autoimmune diseases is a common adverse event. Therefore, use of an IFN-free regimen of direct-acting antivirals for HCV might prevent the extrahepatic manifestation of an immune disorder.

Hepatitis C virus (HCV) has been reported to hijack fatty acid metabolism in infected hepatocytes, taking advantage of lipid droplets for virus assembly. In this study, we analyzed the anti-HCV activity of l-carnitine, a substance involved in the transport of fatty acids into mitochondria. JFH-1 or HCV replicon-transfected Huh7.5.1 cells were treated with or without l-carnitine to examine its anti-HCV effects. The effects of l-carnitine on HCV entry, HCV-induced adipogenesis and lipid droplet formation, and HCV-induced oxidative stress were examined. Treatment of JFH-1-infected cells with l-carnitine inhibited HCV propagation in a concentration-dependent manner. In contrast, l-carnitine had no anti-HCV activity in the HCV replicon system, which is lacking viral assembly. In addition, l-carnitine did not affect HCV entry. However, l-carnitine treatment decreased intracellular lipid droplets, which are crucial for HCV assembly in JFH-1-infected cells. The expression level of CPT-1 was decreased in JFH-1-infected cells, and l-carnitine treatment restored this expression. HCV-infected cells exhibited increased production of reactive oxygen species and glutathione oxidation. l-carnitine decreased oxidative stress induced by JFH-1-infection, as shown by glutathione/glutathione disulfide assays and MitoSOX staining. l-carnitine exhibited anti-HCV activity, possibly by inhibiting HCV assembly and through its anti-adipogenic activity in HCV-infected cells. Moreover, l-carnitine has antioxidant properties in HCV-infected hepatocytes. Overall, these results indicated that l-carnitine may be an effective adjunctive agent in antiviral therapies to treat chronic hepatitis C. J. Med. Virol. 89:857-866, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Hepatitis C virus (HCV)-induced hepatic stress is associated with increased oxidative DNA damage and has been implicated in hepatic inflammation. However, HCV infection and replication are uneven and vary among individual hepatocytes. To investigate the effect of the viral load on host DNA damage, we used an Enhanced Yellow Fluorescent Protein gene (EYFP)-tagged HCV virus to distinguish between HCV intracellular high viral load (HVL) cells and low viral load (LVL) cells. The cell sorting efficiency was confirmed by the high expression of the HCV polyprotein. We found DNA damage γ-H2AX foci in the HVL population. Comet assays demonstrated that HVL was related to the extent of the DNA strand breaks. Surprisingly, the DNA qPCR arrays and western blotting showed that the damage-related genes GPX2, MRE11, phospho-ATM, and OGG1 were significantly up-regulated in LVL cells but inversely down-regulated or consistently expressed in HVL cells. The colony survival assay to examine the repair abilities of these cells in response to irradiation showed that the LVL cells were more resistant to irradiation and had an increased ability to repair radiation-induced damage. This study found that intracellular viral loads drove cellular DNA damage levels but suppressed damage-related gene expression. However, the increase in damage-related gene expression in the LVL cells may be affected by ROS from the HVL cells. These findings provide new insights into the distinct DNA damage and repair responses resulting from different viral loads in HCV-infected cells., Competing Interests: The authors have declared that no competing interests exist.

High rates of sustained virologic response (SVR) has been achieved in Japanese patients with chronic hepatitis C virus (HCV) genotype (GT)1 and GT2 infection treated with ledipasvir/sofosbuvir (LDV/SOF) ±ribavirin (RBV) and SOF+RBV, respectively. We evaluated the effect of baseline HCV NS5A and NS5B resistance-associated variants (RAVs) on treatment outcome and characterized variants at virologic failure. Baseline deep sequencing for NS5A and NS5B genes was performed for all GT1 patients. Deep sequencing of NS5A (GT1 only) and NS5B (GT1 and GT2) was performed for patients who failed treatment or discontinued early with detectable HCV RNA (i.e., >25 IU/mL). In patients with HCV GT1 infection, 22.3% (GT1a: 2/11; GT1b: 74/330) had ≥1 baseline NS5A RAV. The most frequent NS5A RAVs in GT1b were Y93H (17.9%, 59/330) and L31M (2.4%, 8/330). Despite the presence of NS5A RAVs at baseline, 100% and 97% of patients achieved SVR12, compared with 100% and 99% for those with no NS5A RAVs with LDV/SOF and LDV/SOF+RBV, respectively. All patients with NS5B RAVs at baseline achieved SVR12. Of the 153 patients with GT2 infection (GT2a 60.1%, GT2b 39.9%), 3.3% (5/153) experienced viral relapse. No S282T or other NS5B RAVs were detected at baseline or relapse; no change in susceptibility to SOF or RBV was observed at relapse. In conclusion, LDV/SOF and SOF+RBV demonstrate a high barrier to resistance in Japanese patients with HCV GT1 and GT2 infection. The presence of baseline NS5A RAVs did not impact treatment outcome in GT1 Japanese patients treated with LDV/SOF for 12 weeks., (© 2016 John Wiley & Sons Ltd.)

The chaperone system is known to be exploited by viruses for their replication. In the present study, we identified the cochaperone FKBP6 as a host factor required for hepatitis C virus (HCV) replication. FKBP6 is a peptidyl prolyl cis-trans isomerase with three domains of the tetratricopeptide repeat (TPR), but lacks FK-506 binding ability. FKBP6 interacted with HCV nonstructural protein 5A (NS5A) and also formed a complex with FKBP6 itself or FKBP8, which is known to be critical for HCV replication. The Val(121) of NS5A and TPR domains of FKBP6 were responsible for the interaction between NS5A and FKBP6. FKBP6 was colocalized with NS5A, FKBP8, and double-stranded RNA in HCV-infected cells. HCV replication was completely suppressed in FKBP6-knockout hepatoma cell lines, while the expression of FKBP6 restored HCV replication in FKBP6-knockout cells. A treatment with the FKBP8 inhibitor N-(N', N'-dimethylcarboxamidomethyl)cycloheximide impaired the formation of a homo- or hetero-complex consisting of FKBP6 and/or FKBP8, and suppressed HCV replication. HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue. These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A.

Amino acid (aa) polymorphisms in the hepatitis C virus (HCV) genotype 1b core protein have been reported to be a potent predictor for poor response to interferon (IFN)-based therapy and a risk factor for hepatocarcinogenesis. We investigated the effects of these polymorphisms with genotype 1b/2a chimeric viruses that contained polymorphisms of Arg/Gln at aa 70 and Leu/Met at aa 91. We found that infectious virus production was reduced in cells transfected with chimeric virus RNA that had Gln at aa 70 (aa70Q) compared with RNA with Arg at aa 70 (aa70R). Using flow cytometry analysis, we confirmed that HCV core protein accumulated in aa70Q clone transfected cells, and it caused a reduction in cell-surface expression of major histocompatibility complex (MHC) class I molecules induced by IFN treatment through enhanced protein kinase R phosphorylation. We could not detect any effects due to the polymorphism at aa 91. In conclusion, the polymorphism at aa 70 was associated with efficiency of infectious virus production, and this deteriorated virus production in strains with aa70Q resulted in the intracellular accumulation of HCV proteins and attenuation of MHC class I molecule expression. These observations may explain the strain-associated resistance to IFN-based therapy and hepatocarcinogenesis of HCV.

Abstract Hepatitis C virus (HCV) infection is a serious health problem worldwide that can lead to hepatocellular carcinoma or end-stage liver disease. Current treatment with pegylated interferon, ribavirin, and NS3/4A protease inhibitor would lead to a good prognosis in a large population of patients, but there is still no effective vaccine for HCV. HCV robustly infects hepatocytes in the liver. However, extrahepatic manifestations such as mixed cryoglobulinemia, a systemic immune complex-mediated disorder characterized by B-cell proliferation, which may evolve into overt B-cell non-Hodgkin's lymphoma, have been demonstrated. HCV-RNA is often found to be associated with peripheral blood lymphocytes, suggesting a possible interaction with peripheral blood mononuclear cells (PBMCs), especially B-cells with HCV. B-cell HCV infection was a matter of debate for a long time, and the new advance in HCV in vitro infectious systems suggest that exosome can transmit HCV genome to support "infection." We aimed to clarify the susceptibility of primary B-cells to HCV infection, and to study its functional effect. In this article, we found that the recombinant HCV J6JFH1 strain could infect human B-cells isolated from the peripheral blood of normal volunteers by the detection of both HCV-negative-strand RNA by reverse transcription polymerase chain reaction, and NS5A protein. We also show the blocking of HCV replication by type I interferon after B-cell HCV infection. Although HCV replication in B-lymphocytes showed lower efficiency, in comparison with hepatocyte line (Huh7) cells, our results clearly demonstrate that human B-lymphocytes without other non-B-cells can actually be infected with HCV, and that this interaction leads to the induction of B-cells' innate immune response, and change the response of these cells to apoptosis.

Inosine triphosphatase (ITPA) genetic variants are strongly associated with ribavirin (RBV)-induced anaemia during pegylated interferon (PEG-IFN) plus RBV therapy. However, the treatment efficacy of ITPA genetic variants has not been fully explored. We enrolled 309 individuals infected with hepatitis C virus genotype 1, who were treated with PEG-IFN plus RBV for 48 weeks. The ITPA SNP: rs1127354 and IL28B SNP: rs8099917 were genotyped. We examined the risk factors for severe anaemia up to week 12 after the start of treatment and treatment efficacy. The incidence of severe anaemia, ≥ 3 g/dL reduction or <10 g/dL of haemoglobin (Hb) up to week 12, was more frequent in patients with CC at rs1127354 [65% (145/224), 33% (73/224)] than in those with CA/AA [25% (21/85), 6% (8/85)] (P < 0.0001). ITPA genotype, pretreatment Hb level and age were independent predictive factors for severe anaemia: Hb < 10 g/dL. In IL28B favourable type, the sustained virologic response rate was higher in ≥ 60-year-old patients with CA/AA than in those with CC [71% (22/31) vs 40% (26/65), P = 0.005], although there was no significant difference in treatment efficacy according to ITPA genetic variants in the <60-year-old patients. The proportion of patients administered ≥ 80% of the dosage of RBV was significantly higher in the patients with CA/AA than in those with CC (P = 0.025), resulting in a lower relapse rate. In conclusion, ITPA genetic variants were associated with severe RBV-induced anaemia and could influence the efficacy of PEG-IFN plus RBV treatment among elderly patients with IL28B favourable type., (© 2013 John Wiley & Sons Ltd.)

Caffeic acid phenethyl ester (CAPE) has been reported as a multifunctional compound. In this report, we tested the effect of CAPE and its derivatives on hepatitis C virus (HCV) replication in order to develop an effective anti-HCV compound. CAPE and CAPE derivatives exhibited anti-HCV activity against an HCV replicon cell line of genotype 1b with EC50 values in a range from 1.0 to 109.6 µM. Analyses of chemical structure and antiviral activity suggested that the length of the n-alkyl side chain and catechol moiety are responsible for the anti-HCV activity of these compounds. Caffeic acid n-octyl ester exhibited the highest anti-HCV activity among the tested derivatives with an EC50 value of 1.0 µM and an SI value of 63.1 by using the replicon cell line derived from genotype 1b strain Con1. Treatment with caffeic acid n-octyl ester inhibited HCV replication of genotype 2a at a similar level to that of genotype 1b irrespectively of interferon signaling. Caffeic acid n-octyl ester could synergistically enhance the anti-HCV activities of interferon-alpha 2b, daclatasvir, and VX-222, but neither telaprevir nor danoprevir. These results suggest that caffeic acid n-octyl ester is a potential candidate for novel anti-HCV chemotherapy drugs.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that acts as a key player in the HCV replication complex. Understanding the interplay between the viral and cellular components of the HCV replication complex could provide new insight for prevention of the progression of HCV-associated hepatocellular carcinoma (HCC). In this study, the NS5B protein was used as the bait in a pulldown assay to screen for NS5B-interacting proteins that are present in Huh7 hepatoma cell lysates. After mass spectrophotometric analysis, fatty acid synthase (FASN) was found to interact with NS5B. Coimmunoprecipitation and double staining assays further confirmed the direct binding between NS5B and FASN. The domain of NS5B that interacts with FASN was also determined. Moreover, FASN was associated with detergent-resistant lipid rafts and colocalized with NS5B in active HCV replication complexes. In addition, overexpression of FASN enhanced HCV expression in Huh7/Rep-Feo cells, while transfection of FASN small interfering RNA (siRNA) or treatment with FASN-specific inhibitors decreased HCV replication and viral production. Notably, FASN directly increased HCV NS5B RdRp activity in vitro. These results together indicate that FASN interacts with NS5B and modulates HCV replication through a direct increase of NS5B RdRp activity. FASN may thereby serve as a target for the treatment of HCV infection and the prevention of HCV-associated HCC progression.

The non-coding microRNA (miRNA) is involved in the regulation of hepatitis C virus (HCV) infection and offers an alternative target for developing anti-HCV agent. In this study, we aim to identify novel cellular miRNAs that directly target the HCV genome with anti-HCV therapeutic potential. Bioinformatic analyses were performed to unveil liver-abundant miRNAs with predicted target sequences on HCV genome. Various cell-based systems confirmed that let-7b plays a negative role in HCV expression. In particular, let-7b suppressed HCV replicon activity and down-regulated HCV accumulation leading to reduced infectivity of HCVcc. Mutational analysis identified let-7b binding sites at the coding sequences of NS5B and 5'-UTR of HCV genome that were conserved among various HCV genotypes. We further demonstrated that the underlying mechanism for let-7b-mediated suppression of HCV RNA accumulation was not dependent on inhibition of HCV translation. Let-7b and IFNα-2a also elicited a synergistic inhibitory effect on HCV infection. Together, let-7b represents a novel cellular miRNA that targets the HCV genome and elicits anti-HCV activity. This study thereby sheds new insight into understanding the role of host miRNAs in HCV pathogenesis and to developing a potential anti-HCV therapeutic strategy.

Recent studies have demonstrated that the liver-specific microRNA (miRNA) miR-122a plays an important role in the replication of hepatitis C virus (HCV). Antisense nucleotides against miR-122a, including locked nucleic acid (LNA), have shown promising results for suppression of HCV replication; however, a liver-specific delivery system of antisense nucleotides has not been fully developed. In this study, an adenovirus (Ad) vector that expresses tough decoy (TuD)-RNA against miR-122a (TuD-122a) was developed to suppress the HCV replication in the liver hepatocytes. Ad vectors have been well established to exhibit a marked hepatotropism following systemic administration. An in vitro reporter gene expression assay demonstrated that Ad vector-mediated expression of TuD-122a efficiently blocked the miR-122a in Huh-7 cells. Furthermore, transduction with the Ad vector expressing TuD-122a in HCV replicon-expressing cells resulted in significant reduction in the HCV replicon levels. These results indicate that Ad vector-mediated expression of TuD-122a would be a promising tool for treatment of HCV infection., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Hepatitis C virus (HCV) is a causative agent of acute and chronic hepatitis, leading to the development of hepatic cirrhosis and hepatocellular carcinoma. We prepared extracts from 61 marine organisms and screened them by an in vitro fluorescence assay targeting the viral helicase (NS3), which plays an important role in HCV replication, to identify effective candidates for anti-HCV agents. An ethyl acetate-soluble fraction of the feather star Alloeocomatella polycladia exhibited the strongest inhibition of NS3 helicase activity, with an IC(50) of 11.7 µg/mL. The extract of A. polycladia inhibited interaction between NS3 and RNA but not ATPase of NS3. Furthermore, the replication of the replicons derived from three HCV strains of genotype 1b in cultured cells was suppressed by the extract with an EC(50) value of 23 to 44 µg/mL, which is similar to the IC(50) value of the NS3 helicase assay. The extract did not induce interferon or inhibit cell growth. These results suggest that the unknown compound(s) included in A. polycladia can inhibit HCV replication by suppressing the helicase activity of HCV NS3. This study may present a new approach toward the development of a novel therapy for chronic hepatitis C.

Structure-based studies led to the identification of a constrained derivative of S-trityl-l-cysteine (STLC) scaffold as a candidate inhibitor of hepatitis C virus (HCV) NS5B polymerase. A panel of STLC derivatives were synthesized and investigated for their activity against HCV NS5B. Three STLC derivatives, 9, F-3070, and F-3065, were identified as modest HCV NS5B inhibitors with IC(50) values between 22.3 and 39.7 μM. F-3070 and F-3065 displayed potent inhibition of intracellular NS5B activity in the BHK-NS5B-FRLuc reporter and also inhibited HCV RNA replication in the Huh7/Rep-Feo1b reporter system. Binding mode investigations suggested that the STLC scaffold can be used to develop new NS5B inhibitors by further chemical modification at one of the trityl phenyl group., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

To identify novel compounds that possess antiviral activity against hepatitis C virus (HCV), we screened a library of small molecules with various amounts of structural diversity using an HCV replicon-expressing cell line and performed additional validations using the HCV-JFH1 infectious-virus cell culture. Of 4,004 chemical compounds, we identified 4 novel compounds that suppressed HCV replication with 50% effective concentrations of ranging from 0.36 to 4.81 μM. N'-(Morpholine-4-carbonyloxy)-2-(naphthalen-1-yl) acetimidamide (MCNA) was the most potent and also produced a small synergistic effect when used in combination with alpha interferon. Structure-activity relationship (SAR) analyses revealed 4 derivative compounds with antiviral activity. Further SAR analyses revealed that the N-(morpholine-4-carbonyloxy) amidine moiety was a key structural element for antiviral activity. Treatment of cells with MCNA activated nuclear factor κB and downstream gene expression. In conclusion, N-(morpholine-4-carbonyloxy) amidine and other related morpholine compounds specifically suppressed HCV replication and may have potential as novel chemotherapeutic agents.

Unlabelled: Innate immunity plays an important role in host antiviral response to hepatitis C viral (HCV) infection. Recently, single nucleotide polymorphisms (SNPs) of IL28B and host response to peginterferon α (PEG-IFNα) and ribavirin (RBV) were shown to be strongly associated. We aimed to determine the gene expression involving innate immunity in IL28B genotypes and elucidate its relation to response to antiviral treatment. We genotyped IL28B SNPs (rs8099917 and rs12979860) in 88 chronic hepatitis C patients treated with PEG-IFNα-2b/RBV and quantified expressions of viral sensors (RIG-I, MDA5, and LGP2), adaptor molecule (IPS-1), related ubiquitin E3-ligase (RNF125), modulators (ISG15 and USP18), and IL28 (IFNλ). Both IL28B SNPs were 100% identical; 54 patients possessed rs8099917 TT/rs12979860 CC (IL28B major patients) and 34 possessed rs8099917 TG/rs12979860 CT (IL28B minor patients). Hepatic expressions of viral sensors and modulators in IL28B minor patients were significantly up-regulated compared with that in IL28B major patients (≈ 3.3-fold, P < 0.001). However, expression of IPS-1 was significantly lower in IL28B minor patients (1.2-fold, P = 0.028). Expressions of viral sensors and modulators were significantly higher in nonvirological responders (NVR) than that in others despite stratification by IL28B genotype (≈ 2.6-fold, P < 0.001). Multivariate and ROC analyses indicated that higher RIG-I and ISG15 expressions and RIG-I/IPS-1 expression ratio were independent factors for NVR. IPS-1 down-regulation in IL28B minor patients was confirmed by western blotting, and the extent of IPS-1 protein cleavage was associated with the variable treatment response., Conclusion: Gene expression involving innate immunity is strongly associated with IL28B genotype and response to PEG-IFNα/RBV. Both IL28B minor allele and higher RIG-I and ISG15 expressions and RIG-I/IPS-1 ratio are independent factors for NVR., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Combination therapy with ribavirin, interferon, and viral protease inhibitors could be expected to elicit a high level of sustained virologic response in patients infected with hepatitis C virus (HCV). However, several severe side effects of this combination therapy have been encountered in clinical trials. In order to develop more effective and safer anti-HCV compounds, we employed the replicon systems derived from several strains of HCV to screen 84 extracts from 54 organisms that were gathered from the sea surrounding Okinawa Prefecture, Japan. The ethyl acetate-soluble extract that was prepared from marine sponge Amphimedon sp. showed the highest inhibitory effect on viral replication, with EC₅₀ values of 1.5 and 24.9 µg/ml in sub-genomic replicon cell lines derived from genotypes 1b and 2a, respectively. But the extract had no effect on interferon-inducing signaling or cytotoxicity. Treatment with the extract inhibited virus production by 30% relative to the control in the JFH1-Huh7 cell culture system. The in vitro enzymological assays revealed that treatment with the extract suppressed both helicase and protease activities of NS3 with IC₅₀ values of 18.9 and 10.9 µg/ml, respectively. Treatment with the extract of Amphimedon sp. inhibited RNA-binding ability but not ATPase activity. These results suggest that the novel compound(s) included in Amphimedon sp. can target the protease and helicase activities of HCV NS3.

Host tropism of hepatitis C virus (HCV) is limited to human and chimpanzee. HCV infection has never been fully understood because there are few conventional models for HCV infection. Human induced pluripotent stem cell-derived hepatocyte-like (iPS-Hep) cells have been expected to use for drug discovery to predict therapeutic activities and side effects of compounds during the drug discovery process. However, the suitability of iPS-Hep cells as an experimental model for HCV research is not known. Here, we investigated the entry and genomic replication of HCV in iPS-Hep cells by using HCV pseudotype virus (HCVpv) and HCV subgenomic replicons, respectively. We showed that iPS-Hep cells, but not iPS cells, were susceptible to infection with HCVpv. The iPS-Hep cells expressed HCV receptors, including CD81, scavenger receptor class B type I (SR-BI), claudin-1, and occludin; in contrast, the iPS cells showed no expression of SR-BI or claudin-1. HCV RNA genome replication occurred in the iPS-Hep cells. Anti-CD81 antibody, an inhibitor of HCV entry, and interferon, an inhibitor of HCV genomic replication, dose-dependently attenuated HCVpv entry and HCV subgenomic replication in iPS-Hep cells, respectively. These findings suggest that iPS-Hep cells are an appropriate model for HCV infection., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A lack of patient response to alpha interferon (α-IFN) plus ribavirin (RBV) treatment is a major problem in eliminating hepatitis C virus (HCV). We screened chemical libraries for compounds that enhanced cellular responses to α-IFN and identified a triterpenoid, toosendanin (TSN). Here, we studied the effects and mechanisms of action of TSN on HCV replication and its effect on α-IFN signaling. We treated HCV genotype 1b replicon-expressing cells and HCV-J6/JFH-infected cells with TSN, with or without α-IFN, and the level of HCV replication was quantified. To study the effects of TSN on α-IFN signaling, we detected components of the interferon-stimulated gene factor 3 (ISGF3), phosphorylated signal transducer and activator of transcription 1 (STAT1), and STAT2 by Western blotting analysis; expression levels of mRNA of interferon regulatory factor 9 using real-time reverse transcription-PCR (RT-PCR); and interferon-stimulated response element reporter activity and measured the expression levels of interferon-inducible genes for 2',5'-oligoadenylate synthetase, MxA, protein kinase R, and p56 using real-time RT-PCR. TSN alone specifically inhibited expression of the HCV replicon (50% effective concentration = 20.6 nM, 50% cytotoxic concentration > 3 μM, selectivity index > 146). Pretreatment with TSN prior to α-IFN treatment was more effective in suppressing HCV replication than treatment with either drug alone. Although TSN alone did not activate the α-IFN pathway, it significantly enhanced the α-IFN-induced increase of phosphorylated STATs, interferon-stimulated response element activation, and interferon-stimulated gene expression. TSN significantly increased baseline expression of interferon regulatory factor 9, a component of interferon-stimulated gene factor 3. Antiviral effects of treatment with α-IFN can be enhanced by pretreatment with TSN. Its mechanisms of action could potentially be important to identify novel molecular targets to treat HCV infection.

Substitution of amino acids 70 and 91 in the hepatitis C virus (HCV) core region is a significant predictor of poor responses to peginterferon-plus-ribavirin therapy, while their molecular mechanisms remain unclear. Here we investigated these differences in the response to alpha interferon (IFN) by using HCV cell culture with R70Q, R70H, and L91M substitutions. IFN treatment of cells transfected or infected with the wild type or the mutant HCV clones showed that the R70Q, R70H, and L91M core mutants were significantly more resistant than the wild type. Among HCV-transfected cells, intracellular HCV RNA levels were significantly higher for the core mutants than for the wild type, while HCV RNA in culture supernatant was significantly lower for these mutants than for the wild type. IFN-induced phosphorylation of STAT1 and STAT2 and expression of the interferon-inducible genes were significantly lower for the core mutants than for the wild type, suggesting cellular unresponsiveness to IFN. The expression level of an interferon signal attenuator, SOCS3, was significantly higher for the R70Q, R70H, and L91M mutants than for the wild type. Interleukin 6 (IL-6), which upregulates SOCS3, was significantly higher for the R70Q, R70H, and L91M mutants than for the wild type, suggesting interferon resistance, possibly through IL-6-induced, SOCS3-mediated suppression of interferon signaling. Expression levels of endoplasmic reticulum (ER) stress proteins were significantly higher in cells transfected with a core mutant than in those transfected with the wild type. In conclusion, HCV R70 and L91 core mutants were resistant to interferon in vitro, and the resistance may be induced by IL-6-induced upregulation of SOCS3. Those mechanisms may explain clinical interferon resistance of HCV core mutants.

The efficient delivery of the hepatitis C virus (HCV) RNA subgenomic replicon into cells is useful for basic and pharmaceutical studies. The adenovirus (Ad) vector is a convenient and efficient tool for the transduction of foreign genes into cells in vitro and in vivo. However, an Ad vector expressing the HCV replicon has never been developed. In the present study, we developed Ad vector containing an RNA polymerase (pol) I-dependent expression cassette and a tetracycline-controllable RNA pol I-dependent expression system. We prepared a hybrid promoter from the tetracycline-responsive element and the RNA pol I promoter. Ad vector particles coding the hybrid promoter-driven HCV replicon could be amplified, and interferon, an inhibitor of HCV replication, reduced HCV replication in cells transduced with the Ad vector coding HCV replicon. This is the first report of the development of an Ad vector-mediated HCV replicon system.

Evidence from clinical and laboratory studies has accumulated indicating that the activation of the cannabinoid system is crucial for steatosis, especially in non-alcoholic fatty liver disease. However, the association between hepatitis C virus (HCV) infection and the cannabinoid system has not been well investigated and it is unclear whether steatosis in chronic hepatitis C develops via activation of the endocannabinoid/cannabinoid receptor signaling pathway. In this study, we examined the expression of a cannabinoid receptor (CB1) and the lipid accumulation in the hepatic Huh7 cell line, expressing HCV genes. We utilized Huh7/Rep-Feo-1b cells stably expressing HCV non-structural proteins (NS) 3, NS4, NS5A, and NS5B, as well as Tet-On Core-2 cells, in which the HCV core protein expression is inducible. Significantly higher levels of stored triglycerides were found in Huh7/Rep-Feo-1b cells compared to Huh7 cells. Also, triglyceride accumulation and CB1 receptor expression were down-regulated in Huh7/Rep-Feo-1b cells after HCV reduction by IFNα. Moreover, lipid accumulation appeared to increase after CB1 agonist treatment, while it decreased after CB1 antagonist treatment, although significant differences were not found compared to untreated cells. In Tet-On Core-2 cells, induction of HCV core protein expression did not affect CB1 expression or triglyceride accumulation. The results of this study in cultured cells suggest that HCV infection may activate the cannabinoid system and precede steatosis, but the core protein by itself may not have any effect on the cannabinoid system.

The aim of the present study was to clarify the significance of viral factors for pretreatment prediction of sustained virological response to pegylated-interferon (PEG-IFN) plus ribavirin (RBV) therapy for chronic hepatitis C using data mining analysis. Substitutions in the IFN sensitivity-determining region (ISDR) and at position 70 of the HCV core region (Core70) were determined in 505 patients with genotype 1b chronic hepatitis C treated with PEG-IFN plus RBV. Data mining analysis was used to build a predictive model of sustained virological response in patients selected randomly (n = 304). The reproducibility of the model was validated in the remaining 201 patients. Substitutions in ISDR (odds ratio = 9.92, P < 0.0001) and Core70 (odds ratio = 1.92, P = 0.01) predicted sustained virological response independent of other covariates. The decision-tree model revealed that the rate of sustained virological response was highest (83%) in patients with two or more substitutions in ISDR. The overall rate of sustained virological response was 44% in patients with a low number of substitutions in ISDR (0-1) but was 83% in selected subgroups of younger patients (<60 years), wild-type sequence at Core70, and higher level of low-density lipoprotein cholesterol (LDL-C) (≥ 120 mg/dl). Reproducibility of the model was validated (r(2) = 0.94, P < 0.001). In conclusion, substitutions in ISDR and Core70 of HCV are significant predictors of response to PEG-IFN plus RBV therapy. A decision-tree model that includes these viral factors as predictors could identify patients with a high probability of sustained virological response., (Copyright © 2011 Wiley-Liss, Inc.)

Background & Aims: Pegylated interferon and ribavirin (PEG-IFN/RBV) therapy for chronic hepatitis C virus (HCV) genotype 1 infection is effective in 50% of patients. Recent studies revealed an association between the IL28B genotype and treatment response. We aimed to develop a model for the pre-treatment prediction of response using host and viral factors., Methods: Data were collected from 496 patients with HCV genotype 1 treated with PEG-IFN/RBV at five hospitals and universities in Japan. IL28B genotype and mutations in the core and IFN sensitivity determining region (ISDR) of HCV were analyzed to predict response to therapy. The decision model was generated by data mining analysis., Results: The IL28B polymorphism correlated with early virological response and predicted null virological response (NVR) (odds ratio=20.83, p<0.0001) and sustained virological response (SVR) (odds ratio=7.41, p<0.0001) independent of other covariates. Mutations in the ISDR predicted relapse and SVR independent of IL28B. The decision model revealed that patients with the minor IL28B allele and low platelet counts had the highest NVR (84%) and lowest SVR (7%), whereas those with the major IL28B allele and mutations in the ISDR or high platelet counts had the lowest NVR (0-17%) and highest SVR (61-90%). The model had high reproducibility and predicted SVR with 78% specificity and 70% sensitivity., Conclusions: The IL28B polymorphism and mutations in the ISDR of HCV were significant pre-treatment predictors of response to PEG-IFN/RBV. The decision model, including these host and viral factors may support selection of optimum treatment strategy for individual patients., (Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Patients infected with genotype 2b hepatitis C virus (HCV) generally can achieve favorable responses to pegylated-interferon plus ribavirin therapy (PEG-IFN/RBV). However, a proportion of patients show poorer responses and the correlation between viral sequence variation and treatment outcome remains unclear., Methods: The pretreatment complete open reading frame (ORF) sequences of genotype 2b HCV determined by direct sequencing were investigated for correlation with the final outcome in a total of 60 patients., Results: In this study group, 87.5% (14/16) of non-sustained virological response (non-SVR) patients (n = 16) were relapsers. Compared to sustained virological response (SVR) patients (n = 44), non-SVR patients were older and could not achieve prompt viral clearance after the therapy induction. Comparing each viral protein between the two groups, viral sequences were more diverse in SVR patients and that diversity was found primarily in the E1, p7, and NS5A proteins. In searching for specific viral regions associated with the final outcome, several regions in E2, p7, NS2, NS5A, and NS5B were extracted. Among these regions, part of the interferon sensitivity determining region (ISDR) was included. In these regions, amino acid substitutions were associated with the final outcome in an incremental manner, depending upon the number of substitutions., Conclusions: Viral sequences are more diverse in SVR patients than non-SVR patients receiving PEG-IFN/RBV therapy for genotype-2b HCV infection. Through systematic comparison of viral sequences, several specific regions, including part of the ISDR, were extracted as having significant correlation with the final outcome.

Mechanisms of difference in interferon sensitivity between hepatitis C virus (HCV) strains have yet to be clarified. Here, we constructed an infectious genotype2b clone and analyzed differences in interferon-alpha sensitivity between HCV-2b and 2a-JFH1 clones using intergenotypic homologous recombination. The HCV-2b/JFH1 chimeric virus able to infect Huh7.5.1 cells and was significantly more sensitive to IFN than JFH1. IFN-induced expression of MxA and 25-OAS was significantly lower in JFH1 than in 2b/JFH1-infected cells. In JFH1-infected cells, expression of SOCS3 and its inducer, IL-6, was significantly higher than in 2b/JFH1-infected cells. The IFN-resistance of JFH1 cells was negated by siRNA-knock down of SOCS3 expression and by pretreatment with anti-IL6 antibody. In conclusion, intergenotypic differences of IFN sensitivity of HCV may be attributable to the sequences of HCV structural proteins and can be determined by SOCS3 and IL-6 expression levels., (Copyright © 2010 Elsevier Inc. All rights reserved.)

HCV-JFH1 yields subclones that develop cytopathic plaques (Sekine-Osajima Y, et al., Virology 2008; 371:71). Here, we investigated viral amino acid substitutions in cytopathic mutant HCV-JFH1 clones and their characteristics in vitro and in vivo. The mutant viruses with individual C2441S, P2938S or R2985P signature substitutions, and with all three substitutions, showed significantly higher intracellular replication efficiencies and greater cytopathic effects than the parental JFH1 in vitro. The mutant HCV-inoculated mice showed significantly higher serum HCV RNA and higher level of expression of ER stress-related proteins in early period of infection. At 8 weeks post inoculation, these signature mutations had reverted to the wild type sequences. HCV-induced cytopathogenicity is associated with the level of intracellular viral replication and is determined by certain amino acid substitutions in HCV-NS5A and NS5B regions. The cytopathic HCV clones exhibit high replication competence in vivo but may be eliminated during the early stages of infection., (Copyright 2010 Elsevier Inc. All rights reserved.)

Splicing of messenger RNAs is regulated by site-specific binding of members of the serine-arginine-rich (SR) protein family, and SR protein kinases (SRPK) 1 and 2 regulate overall activity of the SR proteins by phosphorylation of their RS domains. We have reported that specifically designed SRPK inhibitors suppressed effectively several DNA and RNA viruses in vitro and in vivo. Here, we show that an SRPK inhibitor, SRPIN340, suppressed in a dose-dependent fashion expression of a hepatitis C virus (HCV) subgenomic replicon and replication of the HCV-JFH1 clone in vitro. The inhibitory effects were not associated with antiproliferative or nonspecific cytotoxic effects on the host cells. Overexpression of SRPK1 or SRPK2 resulted in augmentation of HCV replication, while small interfering RNA (siRNA) knockdown of the SRPKs suppressed HCV replication significantly. Immunocytochemistry showed that SRPKs and the HCV core and NS5A proteins colocalized to some extent in the perinuclear area. Our results demonstrate that SRPKs are host factors essential for HCV replication and that functional inhibitors of these kinases may constitute a new class of antiviral agents against HCV infection.

Background: Pegylated-interferon-alpha 2b (PEG-IFN) plus ribavirin (RBV) therapy is currently the de-facto standard treatment for hepatitis C virus (HCV) infection. The aims of this study were to analyze the clinical and virological factors associated with a higher rate of response in patients with HCV genotype 1b infection treated with combination therapy., Methods: We analyzed, retrospectively, 239 patients with chronic hepatitis C-1b infection who received 48 weeks of combination therapy. We assessed clinical and laboratory parameters, including age, gender, pretreatment hemoglobin, platelet counts, HCV RNA titer, liver histology, the number of interferon sensitivity determining region (ISDR) mutations and substitutions of the core amino acids 70 and 91. Drug adherence was monitored in each patient. We carried out univariate and multivariate statistical analyses of these parameters and clinical responses., Results: On an intention-to-treat (ITT) analysis, 98 of the 239 patients (41%) had sustained virological responses (SVRs). Patients with more than two mutations in the ISDR had significantly higher SVR rates (P<0.01). Univariate analyses showed that stage of fibrosis, hemoglobin, platelet counts, ISDR mutations, serum HCV RNA level, and adherence to PEG-IFN plus RBV were significantly correlated with SVR rates. Multivariate analysis in subjects with good drug adherence extracted the number of ISDR mutations (two or more: odds ratio [OR] 5.181)., Conclusions: The number of mutations in the ISDR sequence of HCV-1b (>or=2) is the most effective parameter predicting a favorable clinical outcome of 48-week PEG-IFN plus RBV therapy in patients with HCV genotype 1b infection.

Background: Hepatitis C virus (HCV) replication is affected by several host factors. Here, we screened host genes and molecular pathways that are involved in HCV replication by comprehensive analyses using two genotypes of HCV replicon-expressing cells, their cured cells and naïve Huh7 cells., Methods: Huh7 cell lines that stably expressed HCV genotype 1b or 2a replicon were used. The cured cells were established by treating HCV replicon cells with interferon-alpha. Expression of 54,675 cellular genes was analyzed by GeneChip DNA microarray. The data were analyzed by using the KEGG Pathway database., Results: Hierarchical clustering analysis showed that the gene-expression profiles of each cell group constituted clear clusters of naïve, HCV replicon-expressed, and cured cell lines. The pathway process analysis between the replicon-expressing and the cured cell lines identified significantly altered pathways, including MAPK, steroid biosynthesis and TGF-beta signaling pathways, suggesting that these pathways were affected directly by HCV replication. Comparison of cured and naïve Huh7 cells identified pathways, including steroid biosynthesis and sphingolipid metabolism, suggesting that these pathways were required for efficient HCV replication. Cytoplasmic lipid droplets were obviously increased in replicon-expressing and cured cells as compared to naïve cells. HCV replication was significantly suppressed by peroxisome proliferator-activated receptor (PPAR)-alpha agonists but augmented by PPAR-gamma agonists., Conclusion: Comprehensive gene expression and pathway analyses show that lipid biosynthesis pathways are crucial to support proficient virus replication. These metabolic pathways could constitute novel antiviral targets against HCV.

A polyphenolic compound from the curry spice turmeric, curcumin, is known to show anti-viral activity against the influenza virus, adenovirus, coxsackievirus, and the human immunodeficiency virus. However, it remains to be determined whether curcumin can inhibit the replication of hepatitis C virus (HCV). In this study, we showed that curcumin decreases HCV gene expression via suppression of the Akt-SREBP-1 activation, not by NF-kappaB pathway. The combination of curcumin and IFNalpha exerted profound inhibitory effects on HCV replication. Collectively, our results indicate that curcumin can suppress HCV replication in vitro and may be potentially useful as novel anti-HCV reagents., (Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Background: Autophagy has been reported to play a pivotal role on the replication of various RNA viruses. In this study, we investigated the role of autophagy on hepatitis C virus (HCV) RNA replication and demonstrated anti-HCV effects of an autophagic proteolysis inhibitor, chloroquine., Methods: Induction of autophagy was evaluated following the transfection of HCV replicon to Huh-7 cells. Next, we investigated the replication of HCV subgenomic replicon in response to treatment with lysosomal protease inhibitors or pharmacological autophagy inhibitor. The effect on HCV replication was analyzed after transfection with siRNA of ATG5, ATG7 and light-chain (LC)-3 to replicon cells. The antiviral effect of chloroquine and/or interferon-alpha (IFNalpha) was evaluated., Results: The transfection of HCV replicon increased the number of autophagosomes to about twofold over untransfected cells. Pharmacological inhibition of autophagic proteolysis significantly suppressed expression level of HCV replicon. Silencing of autophagy-related genes by siRNA transfection significantly blunted the replication of HCV replicon. Treatment of replicon cells with chloroquine suppressed the replication of the HCV replicon in a dose-dependent manner. Furthermore, combination treatment of chloroquine to IFNalpha enhanced the antiviral effect of IFNalpha and prevented re-propagation of HCV replicon. Protein kinase R was activated in cells treated with IFNalpha but not with chloroquine. Incubation with chloroquine decreased degradation of long-lived protein leucine., Conclusion: The results of this study suggest that the replication of HCV replicon utilizes machinery involving cellular autophagic proteolysis. The therapy targeted to autophagic proteolysis by using chloroquine may provide a new therapeutic option against chronic hepatitis C.

Unlabelled: Interferons (IFNs) and the interferon-stimulated genes (ISGs) play a central role in antiviral responses against hepatitis C virus (HCV) infection. We have reported previously that ISGs, including guanylate binding protein 1 (GBP-1), interferon alpha inducible protein (IFI)-6-16, and IFI-27, inhibit HCV subgenomic replication. In this study we investigated the effects of these ISGs against HCV in cell culture and their direct molecular interaction with viral proteins. HCV replication and virus production were suppressed significantly by overexpression of GBP-1, IFI-6-16, or IFI-27. Knockdown of the individual ISGs enhanced HCV RNA replication markedly. A two-hybrid panel of molecular interaction of the ISGs with HCV proteins showed that GBP-1 bound HCV-NS5B directly. A protein truncation assay showed that the guanine binding domain of GBP-1 and the finger domain of NS5B were involved in the interaction. Binding of NS5B with GBP-1 inhibited its guanosine triphosphatase GTPase activity, which is essential for its antiviral effect. Taken together, interferon-induced GBP-1 showed antiviral activity against HCV replication., Conclusion: Binding of the HCV-NS5B protein to GBP-1 countered the antiviral effect by inhibition of its GTPase activity. These mechanisms may contribute to resistance to innate, IFN-mediated antiviral defense and to the clinical persistence of HCV infection.

Chronic hepatitis C virus (HCV) infection is a worldwide public issue. In this study, we performed bioactivity-guided screening of the Lonicera hypoglauca Miq. crude extracts to find for naturally chemical entities with anti-HCV activity. Pheophytin a was identified from the ethanol-soluble fraction of L. hypoglauca that elicited dose-dependent inhibition of HCV viral proteins and RNA expression in both replicon cells and cell culture infectious system. Computational modeling revealed that pheophytin a can bind to the active site of HCV-NS3, suggesting that NS3 is a potent molecular target of pheophytin a. Biochemical analysis further revealed that pheophytin a inhibited NS3 serine protease activity with IC(50)=0.89 microM. Notably, pheophytin a and IFNalpha-2a elicited synergistic anti-HCV activity in replicon cells with no significant cytotoxicity. This study thereby demonstrates for the first time that pheophytin a is a potent HCV-NS3 protease inhibitor and offers insight for development of novel anti-HCV regimens.

Liver diseases associated with hepatitis C virus (HCV) infection have become the major cause of mortality in patients with human immunodeficiency virus (HIV) infection since the introduction of highly active anti-retroviral therapy. HCV-related liver disease is more severe in HIV-infected patients than in non-HIV-infected patients, but the standard therapies used to treat chronic hepatitis C in HCV/HIV coinfected patients are the same as those for patients infected with HCV alone. HIV protease inhibitors might have potential to down-regulate HCV load of HCV/HIV coinfected patients. In this study, we evaluated the effects of nelfinavir on intracellular HCV replication using the HCV replicon system. We constructed an HCV replicon expressing a neomycin-selectable chimeric firefly luciferase reporter protein. Cytotoxicity and apoptosis induced by nelfinavir were assessed and synergism between nelfinavir and interferon (IFN) was calculated using CalcuSyn analysis. Nelfinavir dose-dependently repressed HCV replication at low concentrations (IC(50), 9.88 micromol/L). Nelfinavir failed to induce cytotoxicity or apoptosis at concentrations that inhibited HCV replication. Clinical concentrations of nelfinavir (5 micromol/L) combined with IFN showed synergistic inhibition of HCV replication in our replicon model. Our results suggest that the direct effects of nelfinavir on the HCV subgenome and its synergism with IFN could improve clinical responses to IFN therapy in HCV/HIV coinfected patients.

Cyclosporine A and nonimmunosuppressive cyclophilin (Cyp) inhibitors such as Debio 025, NIM811, and SCY-635 block hepatitis C virus (HCV) replication in vitro. This effect was recently confirmed in HCV-infected patients where Debio 025 treatment dramatically decreased HCV viral load, suggesting that Cyps inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. Recent studies suggest that Cyps are important for HCV replication. However, a profound disagreement currently exists as to the respective roles of Cyp members in HCV replication. In this study, we analyzed the respective contribution of Cyp members to HCV replication by specifically knocking down their expression by both transient and stable small RNA interference. Only the CypA knockdown drastically decreased HCV replication. The re-expression of an exogenous CypA escape protein, which contains escape mutations at the small RNA interference recognition site, restored HCV replication, demonstrating the specificity for the CypA requirement. We then mutated residues that reside in the hydrophobic pocket of CypA where proline-containing peptide substrates and cyclosporine A bind and that are vital for the enzymatic or the hydrophobic pocket binding activity of CypA. Remarkably, these CypA mutants fail to restore HCV replication, suggesting for the first time that HCV exploits either the isomerase or the chaperone activity of CypA to replicate in hepatocytes and that CypA is the principal mediator of the Cyp inhibitor anti-HCV activity. Moreover, we demonstrated that the HCV NS5B polymerase associates with CypA via its enzymatic pocket. The study of the roles of Cyps in HCV replication should lead to the identification of new targets for the development of alternate anti-HCV therapies.

Hepatitis C virus (HCV) chronically infects 3% of the world's population, and complications from HCV are the leading indication for liver transplantation. Given the need for better anti-HCV therapies, one strategy is to identify and target cellular cofactors of the virus lifecycle. Using a genome-wide siRNA library, we identified 96 human genes that support HCV replication, with a significant number of them being involved in vesicle organization and biogenesis. Phosphatidylinositol 4-kinase PI4KA and multiple subunits of the COPI vesicle coat complex were among the genes identified. Consistent with this, pharmacologic inhibitors of COPI and PI4KA blocked HCV replication. Targeting hepcidin, a peptide critical for iron homeostasis, also affected HCV replication, which may explain the known dysregulation of iron homeostasis in HCV infection. The host cofactors for HCV replication identified in this study should serve as a useful resource in delineating new targets for anti-HCV therapies.

Background and Aim: We have reported previously that synthetic small interfering RNA (siRNA) and DNA-based siRNA expression vectors efficiently and specifically suppress hepatitis C virus (HCV) replication in vitro. In this study, we investigated the effects of the siRNA targeting HCV-RNA in vivo., Methods: We constructed recombinant retrovirus and adenovirus expressing short hairpin RNA (shRNA), and transfected into replicon-expressing cells in vitro and transgenic mice in vivo., Results: Retroviral transduction of Huh7 cells to express shRNA and subsequent transfection of an HCV replicon into the cells showed that the cells had acquired resistance to HCV replication. Infection of cells expressing the HCV replicon with an adenovirus expressing shRNA resulted in efficient vector delivery and expression of shRNA, leading to suppression of the replicon in the cells by approximately 10(-3). Intravenous delivery of the adenovirus expressing shRNA into transgenic mice that can be induced to express HCV structural proteins by the Cre/loxP switching system resulted in specific suppression of virus protein synthesis in the liver., Conclusion: Taken together, our results support the feasibility of utilizing gene targeting therapy based on siRNA and/or shRNA expression to counteract HCV replication, which might prove valuable in the treatment of hepatitis C.

Background & Aims: Clinical significance of molecules involving innate immunity in treatment response remains unclear. The aim is to elucidate the mechanisms underlying resistance to antiviral therapy and predictive usefulness of gene quantification in chronic hepatitis C (CH-C)., Methods: We conducted a human study in 74 CH-C patients treated with pegylated interferon alpha-2b and ribavirin and 5 nonviral control patients. Expression of viral sensors, adaptor molecule, related ubiquitin E3-ligase, and modulators were quantified., Results: Hepatic RIG-I, MDA5, LGP2, ISG15, and USP18 in CH-C patients were up-regulated at 2- to 8-fold compared with nonhepatitis C virus patients with a relatively constitutive Cardif. Hepatic RIG-I, MDA5, and LGP2 were significantly up-regulated in nonvirologic responders (NVR) compared with transient (TR) or sustained virologic responders (SVR). Cardif and RNF125 were negatively correlated with RIG-I and significantly suppressed in NVR. Differences among clinical responses in RIG-I/Cardif and RIG-I/RNF125 ratios were conspicuous (NVR/TR/SVR = 1.3:0.6:0.4 and 2.3:1.3:0.8, respectively). Like viral sensors, ISG15 and USP18 were significantly up-regulated in NVR (4-fold and 2.3-fold, respectively). Multivariate and receiver operator characteristic analyses revealed higher RIG-I/Cardif ratio, ISG15, and USP18 predicted NVR. Lower Cardif in NVR was confirmed by its protein level in Western blot. Also, transcriptional responses in peripheral blood mononuclear cells to the therapy were rapid and strong except for Cardif in not only a positive (RIG-I, ISG15, and USP18) but also in a negative regulatory manner (RNF125)., Conclusions: NVR may have adopted a different equilibrium in their innate immune response. High RIG-I/Cardif and RIG-I/RNF125 ratios and ISG15 and USP18 are useful in identifying NVR.