Hepatitis C Virus Cell Culture Research Articles

An immunosuppressive function of interleukin-35 in chronic hepatitis C virus infection

Interleukin (IL)-35, a newly identified member of the IL-12 cytokine family, has been reported to suppress inflammation and induce immunotolerance. However, little is known regarding the role of IL-35 during chronic hepatitis C virus (HCV) infection. Herein, we measured the serum IL-35 concentration of 73 patients with hepatitis C and 22 healthy individuals, as well as further investigated the modulatory function of IL-35 on CD4+CD25+CD127dim/− regulatory T cells (Tregs) and on hepatocytes infected with HCV in cell culture (HCVcc). IL-35 expression was significantly increased in patients with chronic hepatitis C and was positively correlated with the levels of HCV RNA. Inhibition of viral replication led to decreases in the serum levels of IL-35. IL-35 stimulation not only elevated the percentage of Tregs but also robustly inhibited cellular proliferation and up-regulated the production of anti-inflammatory cytokines (e.g., IL-10 and IL-35) in a HCV-specific and non-specific manner, which indicates enhancement of the suppressive function of Tregs. Although IL-35 did not exert anti-HCV activity in HCVcc-infected Huh7.5 cells, it reduced inflammatory cytokine secretion from Huh7.5 cells. This was probably via inhibition of the STAT1 and STAT3 signaling pathways, which could suppress subsequent liver damage due to chronic hepatitis C. The current data suggested that IL-35 contributes to persistent HCV infection by inhibiting antiviral immune activity. Moreover, IL-35 might also protect against HCV-induced liver injury by down-regulating the expression of proinflammatory cytokines. Thus, the immunosuppressive properties of IL-35 might play contradictory roles in maintaining viral persistence and reducing the inflammatory responses in chronic HCV infection.

Efficient Hepatitis C Virus Genotype 1b Core-NS5A Recombinants Permit Efficacy Testing of Protease and NS5A Inhibitors.

Hepatitis C virus (HCV) strains belong to seven genotypes with numerous subtypes that respond differently to antiviral therapies. Genotype 1, and primarily subtype 1b, is the most prevalent genotype worldwide. The development of recombinant HCV infectious cell culture systems for different variants, permitted by the high replication capacity of strain JFH1 (genotype 2a), has advanced efficacy and resistance testing of antivirals. However, efficient infectious JFH1-based cell cultures of subtype 1b are limited and comprise only the 5' untranslated region (5'UTR)-NS2, NS4A, or NS5A regions. Importantly, it has not been possible to develop efficient 1b infectious systems expressing the NS3/4A protease, an important target of direct-acting antivirals. We developed efficient infectious JFH1-based cultures with genotype 1b core-NS5A sequences of strains DH1, Con1, and J4 by using previously identified HCV cell culture adaptive substitutions A1226G, R1496L, and Q1773H. These viruses spread efficiently in Huh7.5 cells by acquiring additional adaptive substitutions, and final recombinants yielded peak supernatant infectivity titers of 4 to 5 log10 focus-forming units (FFU)/ml. We subsequently succeeded in adapting a JFH1-based 5'UTR-NS5A DH1 recombinant to efficient growth in cell culture. We evaluated the efficacy of clinically relevant NS3/4A protease and NS5A inhibitors against the novel genotype 1b viruses, as well as against previously developed 1a viruses. The inhibitors were efficient against all tested genotype 1 viruses, with NS5A inhibitors showing half-maximal effective concentrations several orders of magnitude lower than NS3/4A protease inhibitors. In summary, the developed HCV genotype 1b culture systems represent valuable tools for assessing the efficacy of various classes of antivirals and for other virological studies requiring genotype 1b infectious viruses.

Modulation of Hepatitis C Virus-Specific CD8 Effector T-Cell Function with Antiviral Effect in Infectious Hepatitis C Virus Coculture Model.

The antiviral effects of hepatitis C virus (HCV)-specific CD8 T cells have been shown in an HCV replicon system but not in an authentic infectious HCV cell culture (HCVcc) system. Here, we developed tools to examine the antigenicity of HCV-infected HLA-A2-positive Huh7.5 hepatoma cells (Huh7.5A2 cells) in activating HCV-specific CD8 T cells and the downstream antiviral effects. Infectious HCV epitope mutants encoding the well-defined genotype 1a-derived HLA-A2-restricted HCV NS3-1073 or NS5-2594 epitope were generated from a genotype 2a-derived HCV clone (Jc1Gluc2A) by site-directed mutagenesis. CD8 T-cell lines specific for NS3-1073 and NS5-2594 were expanded from HCV-seropositive persons by peptide stimulation in vitro or engineered from HCV-seronegative donor T cells by transduction of a lentiviral vector expressing HCV-specific T-cell receptors. HCV-specific CD8 T cells were cocultured with Huh7.5 cells that were pulsed with titrating doses of HCV epitope peptides or infected with HCV epitope mutants. HCV-specific CD8 T-cell activation (CD107a, gamma interferon, macrophage inflammatory protein 1β, tumor necrosis factor alpha) was dependent on the peptide concentrations and the relative percentages of HCV-infected Huh7.5A2 cells. HCV-infected Huh7.5A2 cells activated HCV-specific CD8 T cells at levels comparable to those achieved with 0.1 to 2 μM pulsed peptides, providing a novel estimate of the level at which endogenously processed HCV epitopes are presented on HCV-infected cells. While HCV-specific CD8 T-cell activation with cytolytic and antiviral effects was blunted by PD-L1 expression on HCV-infected Huh7.5A2 cells, resulting in the improved viability of Huh7.5A2 cells, PD-1 blockade reversed this effect, producing enhanced cytolytic elimination of HCV-infected Huh7.5A2 cells. Our findings, obtained using an infectious HCVcc system, show that the HCV-specific CD8 T-cell function is modulated by antigen expression levels, the percentage of HCV-infected cells, and the PD-1/PD-L1 pathways and has antiviral and cytotoxic effects.IMPORTANCE We developed several novel molecular and immunological tools to study the interactions among HCV, HCV-infected hepatocytes, and HCV-specific CD8 T cells. Using these tools, we show the level at which HCV-infected hepatoma cells present endogenously processed HCV epitopes to HCV-specific CD8 T cells with antiviral and cytotoxic effects. We also show the marked protective effect of PD-L1 expression on HCV-infected hepatoma cells against HCV-specific CD8 T cells.

How Current Direct-Acting Antiviral and Novel Cell Culture Systems for HCV are Shaping Therapy and Molecular Diagnosis of Chronic HCV Infection?

We have entered a new era of hepatitis C virus (HCV) therapy in which elimination of infection and disease is a real possibility. HCV cell culture models were instrumental for identification of therapeutic targets, testing candidate drugs, and profiling of therapeutic strategies. Here we describe current and novel methods of cell culture systems for HCV that are allowing investigation of HCV life cycle and virus-host interaction required for replication and propagation. The development of protocols to grow infectious virus in culture and generate hepatocyte cell lines from specific individuals hold great promise to investigate the mechanisms exploited by the virus to spread the infection and the host factors critical for HCV replication and propagation, or resistance to infection. Since host factors are presumably conserved and equally interacting with different HCV isolates and genotypes, the development of drugs targeting host factors essential for virus replication holds great promises in further increasing treatment efficacy. Refocusing of therapeutic goals also impacted in vitro diagnosis. The primary goal of anti-HCV therapy is to achieve a sustained virologic response (SVR) defined as "undetectable" HCV RNA genome in the serum or plasma at 12 to 24 weeks following the end of treatment. Use of direct antiviral agents has substantially changed the threshold of the viral load used to define SVR and led to a reassessment, as discussed herein, of result interpretation and requirements of clinically-approved, quantitative molecular assays.

Novel replicons and trans-encapsidation systems for Hepatitis C Virus proteins live imaging and virus-host interaction proteomics

Proteomics and imaging techniques are used more and more in tandem to investigate the virus-host interaction. Herein we present novel replicons, methods and trans-encapsidation systems suitable for determination of Hepatitis C Virus (HCV) proteins interactomes and live imaging of viral proteins dynamics in HCV cell culture (HCVcc) system. To identify endogenous factors involved in the HCV life cycle, we constructed full-length functional replicons with affinity purification (AP) tags fused to NS2 and NS5A proteins. Viral-host interactomes were determined and validated in HCVcc system. To investigate the dynamics of viral-host interactions, we developed a core-inducible packaging cell line which trans-encapsidates various subgenomic replicons suitable for AP in replication and assembly stages. Further, a transient trans-encapsidation system was developed for live imaging of the NS5A viral protein in replication and assembly steps, respectively. The NS5A dynamics was determined also in the full-length HCV replicon system. The analysis of NS5A dynamics showed a decreased mobility of the protein in assembly versus the replication step. The tools presented herein will allow the investigation of HCV-host interaction with improved biological relevance and biosafety.

Amino Acid Mutations in the NS4A Region of Hepatitis C Virus Contribute to Viral Replication and Infectious Virus Production.

The HCV cell culture system using the JFH-1 strain and HuH-7 cells can be used to assess the complete HCV life cycle in cultured cells. This cell culture system has been used to develop direct-acting antivirals against HCV, and the ability to use various HCV strains within this system is important for future studies. In this study, we aimed to establish a novel HCV cell culture system using another HCV genotype 2a strain, J6CF, which replicates in chimpanzees but not in cultured cells. We identified an effective cell culture-adaptive mutation in NS4A and established a replication-competent J6CF strain in cultured cells with minimum essential modifications. The described strategy can be used in establishing a novel HCV cell culture system, and the replication-competent J6CF clone composed of the minimum essential modifications needed for cell culture adaptation will be valuable as another representative of genotype 2a strains.

Pegylated IFN-α suppresses hepatitis C virus by promoting the DAPK-mTOR pathway

Death-associated protein kinase (DAPK) has been found to be induced by IFN, but its antiviral activity remains elusive. Therefore, we investigated whether DAPK plays a role in the pegylated IFN-α (peg-IFN-α)-induced antiviral activity against hepatitis C virus (HCV) replication. Primary human hepatocytes, Huh-7, and infectious HCV cell culture were used to study the relationship between peg-IFN-α and the DAPK-mammalian target of rapamycin (mTOR) pathways. The activation of DAPK and signaling pathways were determined using immunoblotting. By silencing DAPK and mTOR, we further assessed the role of DAPK and mTOR in the peg-IFN-α-induced suppression of HCV replication. Peg-IFN-α up-regulated the expression of DAPK and mTOR, which was associated with the suppression of HCV replication. Overexpression of DAPK enhanced mTOR expression and then inhibited HCV replication. In addition, knockdown of DAPK reduced the expression of mTOR in peg-IFN-α-treated cells, whereas silencing of mTOR had no effect on DAPK expression, suggesting mTOR may be a downstream effector of DAPK. More importantly, knockdown of DAPK or mTOR significantly mitigated the inhibitory effects of peg-IFN-α on HCV replication. In conclusion, our data suggest that the DAPK-mTOR pathway is critical for anti-HCV effects of peg-IFN-α.

Impact of IL-27 on hepatocyte antiviral gene expression and function

Background: Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines. It is a potent cytokine, with potential antiviral impact, and has been shown to play a role in modulating functions of diverse cell types, including Th1, Th2, and NK and B cells, demonstrating both pro- and anti-inflammatory roles. In hepatocytes, it is capable of inducing signal transducer and activator of transcription (STAT)1, STAT3 and interferon-stimulated genes. Methods: To address its role in viral hepatitis, the antiviral activity of IL-27 against hepatitis C virus (HCV) and hepatitis B virus (HBV) was tested in vitro using cell-culture-derived infectious HCV (HCVcc) cell culture system and the HepaRG HBV cell culture model. To further investigate the impact of IL-27 on hepatocytes, Huh7.5 cells were treated with IL-27 to analyse the differentially expressed genes by microarray analysis. Furthermore, by quantitative PCR, we analyzed the up-regulation of chemokine (CXCL)-10 in response to IL-27. Results: In both HCV and HBV infection models, we observed only a modest direct antiviral effect. Microarray analysis showed that the up-regulated genes mostly belonged to antigen presentation and DNA replication pathways, and involved strong up-regulation of CXCL-10, a gene associated with liver inflammation. Overall, gene set enrichment analysis showed a striking correlation of these genes with those up-regulated in response to related cytokines in diverse cell populations. Conclusion: Our data indicate that IL-27 can have a significant pro-inflammatory impact in vitro, although the direct antiviral effect is modest. It may have a potential impact on hepatocyte function, especially chemokine expression and antigen presentation.

Impact of IL-27 on hepatocyte antiviral gene expression and function.

Background: Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines. It is a potent cytokine, with potential antiviral impact, and has been shown to play a role in modulating functions of diverse cell types, including Th1, Th2, and NK and B cells, demonstrating both pro- and anti-inflammatory roles. In hepatocytes, it is capable of inducing signal transducer and activator of transcription (STAT)1, STAT3 and interferon-stimulated genes. Methods: To address its role in viral hepatitis, the antiviral activity of IL-27 against hepatitis C virus (HCV) and hepatitis B virus (HBV) was tested in vitro using cell-culture-derived infectious HCV (HCVcc) cell culture system and the HepaRG HBV cell culture model. To further investigate the impact of IL-27 on hepatocytes, Huh7.5 cells were treated with IL-27 to analyse the differentially expressed genes by microarray analysis. Furthermore, by quantitative PCR, we analyzed the up-regulation of chemokine (CXCL)-10 in response to IL-27. Results: In both HCV and HBV infection models, we observed only a modest direct antiviral effect. Microarray analysis showed that the up-regulated genes mostly belonged to antigen presentation and DNA replication pathways, and involved strong up-regulation of CXCL-10, a gene associated with liver inflammation. Overall, gene set enrichment analysis showed a striking correlation of these genes with those up-regulated in response to related cytokines in diverse cell populations. Conclusion: Our data indicate that IL-27 can have a significant pro-inflammatory impact in vitro, although the direct antiviral effect is modest. It may have a potential impact on hepatocyte function, especially chemokine expression and antigen presentation.

Infection of Hepatocytes With HCV Increases Cell Surface Levels of Heparan Sulfate Proteoglycans, Uptake of Cholesterol and Lipoprotein, and Virus Entry by Up-regulating SMAD6 and SMAD7

The signaling molecule and transcriptional regulator SMAD6, which inhibits the transforming growth factor β signaling pathway, is required for infection of hepatocytes by hepatitis C virus (HCV). We investigated the mechanisms by which SMAD6 and another inhibitory SMAD (SMAD7) promote HCV infection in human hepatoma cells and hepatocytes. We infected Huh7 and Huh7.5.1 cells and primary human hepatocytes with Japanese fulminant hepatitis-1 (JFH1) HCV cell culture system (HCVcc). We then measured HCV binding, intracellular levels of HCV RNA, and expression of target genes. We examined HCV entry in HepG2/microRNA (miR) 122/CD81 cells, which support entry and replication of HCV, were transfected these cells with small interfering RNAs targeting inhibitory SMADs to analyze gene expression profiles. Uptake of labeled low-density lipoprotein (LDL) and cholesterol was measured. Cell surface proteins were quantified by flow cytometry. We obtained liver biopsy samples from 69 patients with chronic HCV infection and 19 uninfected individuals (controls) and measured levels of syndecan 1 (SDC1), SMAD7, and SMAD6 messenger RNAs (mRNAs). Small interfering RNA knockdown of SMAD6 blocked the binding and infection of hepatoma cell lines and primary human hepatocytes by HCV, whereas SMAD6 overexpression increased HCV infection. We found levels of mRNAs encoding heparan sulfate proteoglycans (HSPGs), particularly SDC1 mRNA, and cell surface levels of heparan sulfate to be reduced in cells after SMAD6 knockdown. SMAD6 knockdown also reduced transcription of genes encoding lipoprotein and cholesterol uptake receptors, including the LDL receptor (LDLR), the very LDLR, and the scavenger receptor class B member 1 in hepatocytes; knockdown of SMAD6 also inhibited cell uptake of cholesterol and lipoprotein. Overexpression of SMAD6 increased the expression of these genes. Similar effects were observed with knockdown and overexpression of SMAD7. In addition, HCV infection of cells increased the expression of SMAD6, which required the activity of nuclear factor-κB, but not transforming growth factor β. Liver tissues from patients with chronic HCV infection had significantly higher levels of SMAD6, SMAD7, and HSPG mRNAs than controls. In studies of hepatoma cell lines and primary human hepatocytes, we found that infection with HCV leads to activation of nuclear factor-κB, resulting in increased expression of SMAD6 and SMAD7. Up-regulation of SMAD6 and SMAD7 induces the expression of HSPGs, such as SDC1, as well as LDLR, very LDLR, and the scavenger receptor class B member 1, which promote HCV entry and propagation, as well as cellular uptake of cholesterol and lipoprotein.

Novel functional hepatitis C virus glycoprotein isolates identified using an optimized viral pseudotype entry assay.

Retrovirus pseudotypes are a highly tractable model used to study the entry pathways of enveloped viruses. This model has been extensively applied to the study of the hepatitis C virus (HCV) entry pathway, preclinical screening of antiviral antibodies and for assessing the phenotype of patient-derived viruses using HCV pseudoparticles (HCVpp) possessing the HCV E1 and E2 glycoproteins. However, not all patient-isolated clones produce particles that are infectious in this model. This study investigated factors that might limit phenotyping of patient-isolated HCV glycoproteins. Genetically related HCV glycoproteins from quasispecies in individual patients were discovered to behave very differently in this entry model. Empirical optimization of the ratio of packaging construct and glycoprotein-encoding plasmid was required for successful HCVpp genesis for different clones. The selection of retroviral packaging construct also influenced the function of HCV pseudoparticles. Some glycoprotein constructs tolerated a wide range of assay parameters, while others were much more sensitive to alterations. Furthermore, glycoproteins previously characterized as unable to mediate entry were found to be functional. These findings were validated using chimeric cell-cultured HCV bearing these glycoproteins. Using the same empirical approach we demonstrated that generation of infectious ebolavirus pseudoviruses (EBOVpv) was also sensitive to the amount and ratio of plasmids used, and that protocols for optimal production of these pseudoviruses are dependent on the exact virus glycoprotein construct. These findings demonstrate that it is crucial for studies utilizing pseudoviruses to conduct empirical optimization of pseudotype production for each specific glycoprotein sequence to achieve optimal titres and facilitate accurate phenotyping.

Combination of neutralizing monoclonal antibodies against Hepatitis C virus E2 protein effectively blocks virus infection

Hepatitis C virus (HCV) represents a major global health threat. The envelope glycoproteins, E1-E2 of HCV play an important role in infection by binding to hepatocyte surface receptors leading to viral entry. Several regions on the E1-E2 are conserved for maintaining structural stability, despite the high mutation rate of HCV. Identification of antigenic determinants in these domains would aid in the development of anti-virals. The present study was aimed to delineate neutralizing epitopes by generating monoclonal antibodies (mAbs) to envelope proteins that can block virus binding and entry. Using HCV-like particles (HCV-LPs) corresponding to genotype 3a (prevalent in India), we obtained three mAbs specific for the E2 protein that significantly inhibited virus binding to hepatoma cells. Using overlapping protein fragments and peptides of the E2 protein, the epitopes corresponding to the mAbs were delineated. MAbs H6D3 and A10F2 recognise sequential linear epitopes, whereas, mAb E3D8 recognises a discontinuous epitope. The epitope of mAb E3D8 overlaps with the CD81 receptor-binding site and that of mAb A10F2 with the hypervariable region 2 of the E2 protein. The epitopes corresponding to these mAbs are distinct and unique. A combination of these antibodies significantly inhibited HCV binding and entry in both HCV pseudoparticle (in vitro) and HCV cell culture (ex vivo) system compared to the mAbs alone (P<0.0001). In conclusion, our findings support the potential of employing a cocktail of neutralizing mAbs in the management of HCV infection.

Fungus-Derived Neoechinulin B as a Novel Antagonist of Liver X Receptor, Identified by Chemical Genetics Using a Hepatitis C Virus Cell Culture System.

Cell culture systems reproducing virus replication can serve as unique models for the discovery of novel bioactive molecules. Here, using a hepatitis C virus (HCV) cell culture system, we identified neoechinulin B (NeoB), a fungus-derived compound, as an inhibitor of the liver X receptor (LXR). NeoB was initially identified by chemical screening as a compound that impeded the production of infectious HCV. Genome-wide transcriptome analysis and reporter assays revealed that NeoB specifically inhibits LXR-mediated transcription. NeoB was also shown to interact directly with LXRs. Analysis of structural analogs suggested that the molecular interaction of NeoB with LXR correlated with the capacity to inactivate LXR-mediated transcription and to modulate lipid metabolism in hepatocytes. Our data strongly suggested that NeoB is a novel LXR antagonist. Analysis using NeoB as a bioprobe revealed that LXRs support HCV replication: LXR inactivation resulted in dispersion of double-membrane vesicles, putative viral replication sites. Indeed, cells treated with NeoB showed decreased replicative permissiveness for poliovirus, which also replicates in double-membrane vesicles, but not for dengue virus, which replicates via a distinct membrane compartment. Together, our data suggest that LXR-mediated transcription regulates the formation of virus-associated membrane compartments. Significantly, inhibition of LXRs by NeoB enhanced the activity of all known classes of anti-HCV agents, and NeoB showed especially strong synergy when combined with interferon or an HCV NS5A inhibitor. Thus, our chemical genetics analysis demonstrates the utility of the HCV cell culture system for identifying novel bioactive molecules and characterizing the virus-host interaction machinery. Hepatitis C virus (HCV) is highly dependent on host factors for efficient replication. In the present study, we used an HCV cell culture system to screen an uncharacterized chemical library. Our results identified neoechinulin B (NeoB) as a novel inhibitor of the liver X receptor (LXR). NeoB inhibited the induction of LXR-regulated genes and altered lipid metabolism. Intriguingly, our results indicated that LXRs are critical to the process of HCV replication: LXR inactivation by NeoB disrupted double-membrane vesicles, putative sites of viral replication. Moreover, NeoB augmented the antiviral activity of all known classes of currently approved anti-HCV agents without increasing cytotoxicity. Thus, our strategy directly links the identification of novel bioactive compounds to basic virology and the development of new antiviral agents.

Completion of the Entire Hepatitis C Virus Life Cycle in Vero Cells Derived from Monkey Kidney.

ABSTRACTA hepatitis C virus (HCV) cell culture system incorporating the JFH-1 strain and the human hepatoma cell line HuH-7 enabled the production of infectious HCV particles. Several host factors were identified as essential for HCV replication. Supplementation of these factors in nonhepatic human cell lines enabled HCV replication and particle production. Vero cells established from monkey kidney are commonly used for the production of vaccines against a variety of viruses. In this study, we aimed to establish a novel Vero cell line to reconstruct the HCV life cycle. Unmodified Vero cells did not allow HCV infection or replication. The expression of microRNA 122 (miR-122), an essential factor for HCV replication, is notably low in Vero cells. Therefore, we supplemented Vero cells with miR-122 and found that HCV replication was enhanced. However, Vero cells that expressed miR-122 still did not allow HCV infection. We supplemented HCV receptor molecules and found that scavenger receptor class B type I (SRBI) was essential for HCV infection in Vero cells. The supplementation of apolipoprotein E (ApoE), a host factor important for virus production, enabled the production of infectious virus in Vero cells. Finally, we created a Vero cell line that expressed the essential factors miR-122, SRBI, and ApoE; the entire HCV life cycle, including infection, replication, and infectious virus production, was completed in these cells. In conclusion, we demonstrated that miR-122, SRBI, and ApoE were necessary and sufficient for the completion of the entire HCV life cycle in nonhuman, nonhepatic Vero cells.

Role of Conserved E2 Residue W420 in Receptor Binding and Hepatitis C Virus Infection.

Hepatitis C virus (HCV) enters cells via interactions with several host factors, a key one being that between the viral E2 envelope glycoprotein and the CD81 receptor. We previously identified E2 tryptophan residue 420 (W420) as an essential CD81-binding residue. However, the importance of W420 in the context of the native virion is unknown, as those previous studies predate the infectious HCV cell culture (cell culture-derived HCV [HCVcc]) system. Here, we introduced four separate mutations (F, Y, A, or R) at position 420 within the infectious HCVcc JFH-1 genome and characterized their effects on the viral life cycle. While all mutations reduced E2-CD81 binding, only two (W420A and W420R) reduced HCVcc infectivity. Further analyses of mutants with hydrophobic residues (F or Y) found that interactions with the receptors SR-BI and CD81 were modulated, which in turn determined the viral uptake route. Both mutant viruses were significantly less dependent on SR-BI, and its lipid transfer activity, for virus entry. Furthermore, these viruses were resistant to the drug erlotinib, which targets epidermal growth factor receptor (EGFR) (a host cofactor for HCV entry) and also blocks SR-BI-dependent high-density lipoprotein (HDL)-mediated enhancement of virus entry. Together, our data indicate a model where an alteration at position 420 causes a subtle change in the E2 conformation that prevents interaction with SR-BI and increases accessibility to the CD81-binding site, in turn favoring a particular internalization route. These results further show that a hydrophobic residue with a strong preference for tryptophan at position 420 is important, both functionally and structurally, to provide an additional hydrophobic anchor to stabilize the E2-CD81 interaction. Hepatitis C virus (HCV) is a leading cause of liver disease, causing up to 500,000 deaths annually. The first step in the viral life cycle is the entry process. This study investigates the role of a highly conserved residue, tryptophan residue 420, of the viral glycoprotein E2 in this process. We analyzed the effect of changing this residue in the virus and confirmed that this region is important for binding to the CD81 receptor. Furthermore, alteration of this residue modulated interactions with the SR-BI receptor, and changes to these key interactions were found to affect the virus internalization route involving the host cofactor EGFR. Our results also show that the nature of the amino acid at this position is important functionally and structurally to provide an anchor point to stabilize the E2-CD81 interaction.

Activity-based and fraction-guided analysis of Phyllanthus urinaria identifies loliolide as a potent inhibitor of hepatitis C virus entry

Without a vaccine, hepatitis C virus (HCV) remains a global medical and socio-economic burden, predisposing about 170 million carriers worldwide to end-stage liver diseases including cirrhosis and hepatocellular carcinoma. Although the recently developed direct-acting antivirals (DAAs) have revolutionized hepatitis C treatment, most of them are unsuitable for monotherapy due to risks of resistance, thus necessitating combination with interferon (IFN)-alpha, ribavirin, or additional DAAs. More importantly, the high cost associated with the DAAs restricts their accessibility to most parts of the world. Developing novel cost-effective anti-HCV therapeutics may help expand the scope of antivirals and treatment strategies against hepatitis C. Herein, we applied an activity-based and fraction-guided analysis of extracts from the medicinal plant Phyllanthus urinaria (P. urinaria), which yielded fraction 13 (F13) as possessing the most potent inhibitory activity against early viral entry of cell-culture HCV infection. Chemical analysis (silica gel chromatography followed by ESI LC-MS plus 1H and 13C NMR) of F13 identified loliolide (LOD), a monoterpenoid lactone, as a novel inhibitor of HCV entry. Specifically, LOD could efficiently inactivate HCV free virus particles, abrogate viral attachment, and impede viral entry/fusion, with minimal effect on viral replication/translation, particle production, and induction of type I IFN host antiviral immune response. ELISA-based binding analysis confirmed the monoterpenoid's ability in efficiently blocking HCV particle attachment to the host cell surface. Furthermore, LOD could inhibit infection by several genotypic strains of HCV. This is the first report characterizing P. urinaria and its bioactive compound LOD as potent HCV entry inhibitors, which merit further evaluation for development as candidate antiviral agents against hepatitis C.

Evaluation of Interferon Resistance in Newly Established Genotype 1b Hepatitis C Virus Cell Culture System.

Background and Aims: The hepatitis C virus (HCV) genotype 1b is known to exhibit treatment resistance with respect to interferon (IFN) therapy. Substitution of amino acids 70 and 91 in the core region of the 1b genotype is a significant predictor of liver carcinogenesis and poor response to pegylated-IFN-α and ribavirin therapy. However, the molecular mechanism has not yet been clearly elucidated because of limitations of the HCV genotype 1b infectious model. Recently, the TPF1-M170T HCV genotype 1b cell culture system was established, in which the clone successfully replicates and infects Huh-7-derived Huh7-ALS32.50 cells. Therefore, the purpose of this study was to compare IFN resistance in various HCV clones using this system. Methods: HCV core amino acid substitutions R70Q and L91M were introduced to the TPF1-M170T clone and then transfected into Huh7-ALS32.50 cells. To evaluate the production of each virus, intracellular HCV core antigens were measured. Results were confirmed with Western blot analysis using anti-NS5A antibodies, and IFN sensitivity was subsequently measured. Results: Each clone was transfected successfully compared with JFH-1, with a significant difference in intracellular HCV core antigen (p < 0.05), an indicator of continuous HCV replication. Among all clones, L91M showed the highest increase in the HCV core antigen and HCV protein. There was no significant resistance against IFN treatment in core substitutions; however, IFN sensitivity was significantly different between the wildtype core and JFH-1 (p < 0.05). Conclusions: A novel genotype 1b HCV cell culture was constructed with core amino acid substitutions, which demonstrated IFN resistance of genotype 1b. This system will be useful for future analyses into the mechanisms of HCV genotype 1b treatment.

MEAN inhibits hepatitis C virus replication by interfering with a polypyrimidine tract-binding protein.

MEAN (6‐methoxyethylamino‐numonafide) is a small molecule compound, and here, we report that it effectively inhibits hepatitis C virus (HCV) infection in an HCV cell culture system using a JC1‐Luc chimeric virus, with a 50% effective concentration (EC50) of 2.36 ± 0.29 μM. Drug combination usage analyses demonstrated that MEAN was synergistic with interferon α, ITX5061 and ribavirin. In addition, MEAN effectively inhibits N415D mutant virus and G451R mutant viral infections. Mechanistic studies show that the treatment of HCV‐infected hepatocytes with MEAN inhibits HCV replication but not translation. Furthermore, treatment with MEAN significantly reduces polypyrimidine tract‐binding protein (PTB) levels and blocks the cytoplasmic redistribution of PTB upon infection. In the host cytoplasm, PTB is directly associated with HCV replication, and the inhibition of HCV replication by MEAN can result in the sequestration of PTB in treated nuclei. Taken together, these results indicate that MEAN is a potential therapeutic candidate for HCV infection, and the targeting of the nucleo‐cytoplasmic translocation of the host PTB protein could be a novel strategy to interrupt HCV replication.

Cellular Mechanism for Impaired Hepatitis C Virus Clearance by Interferon Associated with IFNL3 Gene Polymorphisms Relates to Intrahepatic Interferon-λ Expression

The single nucleotide polymorphism located within the IFNL3 (also known as IL28B) promoter is one of the host factors associated with hepatitis C virus (HCV) clearance by interferon (IFN)-α therapy; however the mechanism remains unknown. We investigated how IL28B gene polymorphism influences HCV clearance with infected primary human hepatocytes, liver biopsies, and hepatoma cell lines. Our study confirms that the rs12979860-T/T genotype has a strong correlation with ss469415590-ΔG/ΔG single nucleotide polymorphism that produces IFN-λ4 protein. We found that IFN-α and IFN-λ1 antiviral activity against HCV was impaired in IL28B T/T infected hepatocytes compared with C/C genotype. Western blot analysis showed that IL28B TT genotype hepatocytes expressed higher levels of IFN-λ proteins (IL28B, IL-29), preactivated IFN-stimulated gene (ISG) expression, and impaired Stat phosphorylation when stimulated with either IFN-α or IFN-λ1. Furthermore, we showed that silencing IFN-λ1 in T/T cell line reduced basal ISG expression and improved antiviral activity. Likewise, overexpression of IFN-λ (1 to 4) in C/C cells induced basal ISG expression and prevented IFN-α antiviral activity. We showed that IFN-λ4, produced at low level only in T/T cells induced expression of IL28B and IL-29 and prevented IFN-α antiviral activity in HCV cell culture. Our results suggest that IFN-λ4 protein expression associated with the IL28B-T/T variant preactivates the Janus kinase-Stat signaling, leading to impaired HCV clearance by both IFN-α and IFN-λ.

Benzothiazepinecarboxamides: Novel hepatitis C virus inhibitors that interfere with viral entry and the generation of infectious virions

Upon screening synthetic small molecule libraries with the infectious hepatitis C virus (HCV) cell culture system, we identified a benzothiazepinecarboxamide (BTC) scaffold that inhibits HCV. A structure-activity relationship (SAR) study with BTCs was performed, and modifications that led to nanomolar antiviral activity and improved the selective index (CC50/EC50) by more than 1000-fold were identified. In addition, a pharmacophore modeling study determined that the tricyclic core and positive charge on the piperidine moiety were essential for antiviral activity. Furthermore, we demonstrated that BTC interferes with HCV glycoprotein E1/E2-mediated viral entry and the generation of infectious virions by using HCV pseudoparticle and cell culture supernatant transfer assays, respectively. BTC showed potent antiviral activity against HCV genotype 2 (EC50 = 0.01 ± 0.01 μM), but was less potent against a genotype 1/2 chimeric virus (EC50 = 2.71 ± 0.05 μM), which expressed the structural proteins of HCV genotype 1. In summary, we identified, optimized, and characterized novel BTC inhibitors that interfere with early and late steps of the HCV viral life cycle.