Japanese Fulminant Hepatitis Research Articles

Anti-Hepatitis C Virus Activity of Various Indonesian Plants from Balikpapan Botanical Garden, East Borneo

Background: Hepatitis C Virus infection is a serious health problem that leads to chronic liver disease, liver cirrhosis, hepatocellular carcinoma, which causes high morbidity. Direct-Acting Antiviral Agents have been used as anti-hepatitis C Virus therapy. However, it was covered only in limited patients due to the high cost. Moreover, serious side effects and resistance cases were also reported in some HCV genotypes. Objective: This research aimed to find new anti-HCV from some Indonesia plants collected from Balikpapan Botanical Garden, East Borneo. Methods: Twenty-one leaf and stem barks extracts were successively extracted in n-hexane, dichloromethane, and methanol. Extracts were screened for their anti-HCV activity under in vitro culture cells in the concentration of 30 µg/mL. Plant extracts were inoculated in the Human Hepatocellular 7it and infected with HCV Japanese Fulminant Hepatitis strain 1a. Determination of 50% Inhibitory Concentration (IC50) value was further conducted at concentration of 100; 30; 10; 1; 0.1; 0.01 µg/ml of extracts. Results: In vitro anti-HCV activity revealed that among 21 plants extract, 11 extracts, namely, n-hexane extract from Luvunga scandens leaves, DCM extract from the leaf of L. scandens, Artocarpus sericicarpus, Artocarpus dadah, Eusideroxylon zwageri, Neolitsea cassiaefolia, methanol extract from A. sericicarpus and A. anisophyllus leaves, DCM extract from A. anisophyllus and A. elmeri stem bark, methanol extract from A. dadah stem bark, having potential inhibition with IC50 range 0.08 ± 0.05 to 12.01 ± 0.95 µg/mL. Conclusions: These results indicate that the eleven extracts could be good candidates as sources of anti-HCV agents.

Reciprocal induction of hepatitis C virus replication and stimulation of hepatic profibrogenic cytokine release and cellular viability by YKL-40.

BackgroundPrevious studies have suggested the YKL-40/chitinase 3-like protein 1 protein is upregulated in chronic hepatitis C virus (HCV) infection with fibrosis. We sought to determine whether HCV regulates YKL-40 expression and to elucidate the mechanisms by which YKL-40 mediates the liver fibrosis caused by HCV infection.MethodsWe used purified protein, small molecule inhibitors and short-interfering RNAs to over-express or knock down certain kinases to explore the mechanisms underlying the regulation by HCV of YKL-40 expression in the Japanese fulminant hepatitis 1 (JFH1) model.ResultsHCV induced YKL-40 production in hepatic parenchymal cells. Further, HCV-mediated upregulation of YKL-40 through cooperative induction of tumor necrosis factor-α (TNF-α) and reactive oxygen species (ROS)-mitogen-activated protein kinase MAPKs, which are nuclear factor-κB (NF-κB)-dependent pathways. YKL-40 protein also mildly increased HCV replication, triggering hepatic profibrogenic cytokine release and cellular viability as feedback. Additionally, hepatic parenchymal cells were the sole source of YKL-40 production in the infectious JFH1 model, whereas YKL-40 was under-detected in hepatic stellate cells (HSCs) in the presence or absence of the JFH1 supernatant, which was not investigated so far.ConclusionsHCV induced and maintained secretion of YKL-40 through sustained activation of NF-κB via cooperative induction of the TNF-α and ROS-MAPKs pathways. HCV interacted with YKL-40 to enhance the progression of hepatic fibrosis. These findings support a potential role for blockade of YKL-40 as an antifibrotic strategy.

A Long Noncoding RNA Regulates Hepatitis C Virus Infection Through Interferon Alpha-Inducible Protein 6.

Long noncoding RNAs (lncRNAs) play a critical role in the regulation of many important cellular processes. However, the mechanisms by which lncRNAs regulate viral infection and host immune responses are not well understood. We sought to explore lncRNA regulation of hepatitis C virus (HCV) infection and interferon response. We performed RNA sequencing (RNAseq) in Huh7.5.1 cells with or without interferon alpha (IFNα) treatment. Clustered regularly interspaced short palindromic repeats/Cas9 guide RNA (gRNA) was used to knock out selected genes. The promoter clones were constructed, and the activity of related interferon-stimulated genes (ISGs) were detected by the secrete-pair dual luminescence assay. We constructed the full-length and four deletion mutants of an interferon-induced lncRNA RP11-288L9.4 (lncRNA-IFI6) based on predicted secondary structure. Selected gene mRNAs and their proteins, together with HCV infection, in Huh7.5.1 cells and primary human hepatocytes (PHHs) were monitored by quantitative real-time PCR (qRT-PCR) and western blot. We obtained 7,901 lncRNAs from RNAseq. A total of 1,062 host-encoded lncRNAs were significantly differentially regulated by IFNα treatment. We found that lncRNA-IFI6 gRNA significantly inhibited HCV infection compared with negative gRNA control. The expression of the antiviral ISG IFI6 was significantly increased following lncRNA-IFI6 gRNA editing compared with negative gRNA control in Japanese fulminant hepatitis 1 (JFH1)-infected Huh7.5.1 cells and PHHs. We observed that lncRNA-IFI6 regulation of HCV was independent of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling. lncRNA-IFI6 negatively regulated IFI6 promoter function through histone modification. Overexpression of the truncated spatial domain or full-length lncRNA-IFI6 inhibited IFI6 expression and increased HCV replication. Conclusion: A lncRNA, lncRNA-IFI6, regulates antiviral innate immunity in the JFH1 HCV infection model. lncRNA-IFI6 regulates HCV infection independently of the JAK-STAT pathway. lncRNA-IFI6 exerts its regulatory function via promoter activation and histone modification of IFI6 through its spatial domain.

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.

Design, Construction and Evaluation of 1a/JFH1 HCV Chimera by Replacing the Intergenotypic Variable Region.

BackgroundThe E2 glycoprotein is an important encoded hepatitis C virus (HCV) protein that contains three different variable regions.ObjectivesThe aim of the present study was to construct an HCV 1a/JFH1 chimeric virus by replacing the intergenotypic variable region (igVR) fragment of the highly variable region of the E2 gene of the Japanese Fulminant hepatitis genotype 2a JFH1 virus with a similar region of HCV genotype 1a. This chimera was produced as a model virus with the ability to be cultured. We analyzed the adapted virus and the variations of nucleic acids within it.MethodsSpecific primers were designed for the igVR of HCV genotype 1a followed by the overlap-PCR method for the synthesis of the desired DNA fragment. The amplified igVR-1a chimera gene and pFL-J6/JFH were digested by KpnI and BsiWI restriction enzymes, and the fragment was ligated into pFL-J6/JFH. The recombinant vector was transformed into Escherichia coli JM109 strain competent cells. All clones were confirmed by colony PCR using specific primers, and the confirmed recombinant vector was sequenced. The recombinant vector was targeted for RNA synthesis by T7 RNA polymerase enzyme. RNA transfection was performed in the Huh7.5 cell line. Virus production in several passages and the evaluated viral load were studied using quantitative real-time PCR and ELISA methods. After 30 passages, the RNA virus was extracted and cloned in PCDNA3.1 vector, and was then sequencedResultsQuantitative real-time PCR results showed 11,292,514 copies/mL of chimeric virus production in cell culture. The virus production was confirmed using ELISA, which showed a virus core production of 808.2 pg/mL. The results of cloning and sequencing showed that some of the nucleic acids in the chimera virus were changed, affecting the viral behavior in the cell culture.ConclusionsReal-time PCR and ELISA showed high levels of production of 1a/JFH1 chimeric HCV in the Huh7.5 cell culture. The constructed virus can be used for future studies, including the development of new HCV drugs and vaccines.

Hepatitis C Virus-Induced Monocyte Differentiation Into Polarized M2 Macrophages Promotes Stellate Cell Activation via TGF-β

Background & AimsMonocyte and macrophage (MΦ) activation contributes to the pathogenesis of chronic hepatitis C virus (HCV) infection. Disease pathogenesis is regulated by both liver-resident MΦs and monocytes recruited as precursors of MΦs into the damaged liver. Monocytes differentiate into M1 (classic/proinflammatory) or M2 (alternative/anti-inflammatory) polarized MΦs in response to tissue microenvironment. We hypothesized that HCV-infected hepatoma cells (infected with Japanese fulminant hepatitis-1 [Huh7.5/JFH-1]) induce monocyte differentiation into polarized MΦs.MethodsHealthy human monocytes were co-cultured with Huh7.5/JFH-1 cells or cell-free virus for 7 days and analyzed for MΦ markers and cytokine levels. A similar analysis was performed on circulating monocytes and liver MΦs from HCV-infected patients and controls.ResultsHuh7.5/JFH-1 cells induced monocytes to differentiate into MΦs with increased expression of CD14 and CD68. HCV-MΦs showed M2 surface markers (CD206, CD163, and Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN)) and produced both proinflammatory and anti-inflammatory cytokines. HCV-induced early interleukin 1β production promoted transforming growth factor (TGF)β production and MΦ polarization to an M2 phenotype. TGF-β secreted by M2-MΦ led to hepatic stellate cell activation indicated by increased expression of collagen, tissue inhibitor of metalloproteinase 1, and α-smooth muscle actin. In vivo, we observed a significant increase in M2 marker (CD206) expression on circulating monocytes and in the liver of chronic HCV-infected patients. Furthermore, we observed the presence of a unique collagen-expressing CD14+CD206+ monocyte population in HCV patients that correlated with liver fibrosis.ConclusionsWe show an important role for HCV in induction of monocyte differentiation into MΦs with a mixed M1/M2 cytokine profile and M2 surface phenotype that promote stellate cell activation via TGF-β. We also identified circulating monocytes expressing M2 marker and collagen in chronic HCV infection that can be explored as a biomarker.

Bioinformatics analysis of hepatitis C virus genotype 2a-induced human hepatocellular carcinoma in Huh7 cells

Hepatocellular carcinoma (HCC) is a liver cancer that could be induced by hepatitis C virus genotype 2a Japanese fulminant hepatitis-1 (JFH-1) strain. The aim of this study was to investigate the molecular mechanisms of HCC. The microarray data GSE20948 includes 14 JFH-1- and 14 mock (equal volume of medium [control])-infected Huh7 samples. The data were downloaded from the Gene Expression Omnibus. After data processing, soft cluster analyses were performed to identify co-regulated genes with similar temporal expression patterns. Functional and pathway enrichment analyses, as well as functional annotation analysis, were performed. Subsequently, combined networks of protein–protein interaction network, microRNA regulatory network, and transcriptional regulatory network were constructed. Hub nodes, modules, and five clusters of co-regulated genes were also identified. In total, 173 up and 207 down co-regulated genes were separately identified in JFH-1-infected Huh7 cells compared with those of control cells. Functional enrichment analysis indicated that up co-regulated genes were related to skeletal system morphogenesis and neuron differentiation and down co-regulated genes were related to steroid/cholesterol/sterol metabolisms. Hub genes (such as IRF1, GBP1, ICAM1, Foxa1, DHCR7, HMGCS2, and MSMO1) were identified. Transcription factors IRF1 and Foxa1 were the targets of miR-130a, miR-17-5p, and miR-20a. PPARGC1A was targeted by miR-29 family, and MSMO1 was the target of miR-23 family. Hub nodes (such as IRF1, GBP1, ICAM1, Foxa1, DHCR7, HMGCS2, and MSMO1) and microRNAs might be used as candidate biomarkers of JFH-1-infected HCC.

Selection of a hepatitis C virus with altered entry factor requirements reveals a genetic interaction between the E1 glycoprotein and claudins.

Hepatitis C virus (HCV) cell entry is a complex, multistep process requiring numerous host cell factors, including the tight junction protein claudin-1 (CLDN1). It is not known whether CLDN1 and the HCV glycoproteins physically interact. Therefore, the focus of this work was to study genetic interactions between CLDN1 and HCV. We used CRISPR technology to generate CLDN1 knockout (KO) Huh-7.5 cells, which could not be infected by genotype 2a Jc1 HCV unless CLDN1 expression was restored. Passage of Jc1-transfected CLDN1 KO cells resulted in the selection of a virus that could infect these cells. This virus encoded a single mutation, H316N (numbered relative to the HCV polyprotein), in the E1 glycoprotein. Whereas Jc1 H316N efficiently infected cells lacking CLDN1, such infection was blocked by an antibody targeting CLDN6, another member of the claudin family that is expressed in these cells. Furthermore, HuH6 cells, which express CLDN6, but not CLDN1, were infectable only with the mutant virus. Thus, this mutant virus adapted to the loss of CLDN1 by developing the capacity to utilize other CLDNs. Indeed, CLDN1/CLDN6 double-KO Huh-7.5 cells supported infection by the mutant virus only when CLDN1, CLDN6, or CLDN9 was expressed. Finally, this phenotype was not genotype dependent, given that the H316N mutation rendered a Japanese fulminant hepatitis 1 chimeric HCV genome encoding the genotype 5a glycoproteins able to utilize CLDN6 for host cell entry. These data demonstrate plasticity of HCV virus-host interactions, where a previously CLDN1-dependent virus was capable of evolving to use CLDN6. They also reveal a role for E1 in determining entry factor usage and imply a direct, physical interaction between E1 and CLDNs.

Efficient virus assembly, but not infectivity, determines the magnitude of hepatitis C virus-induced interferon alpha responses of plasmacytoid dendritic cells.

Worldwide, approximately 160 million people are chronically infected with hepatitis C virus (HCV), seven distinct genotypes of which are discriminated. The hallmarks of HCV are its genetic variability and the divergent courses of hepatitis C progression in patients. We assessed whether intragenotypic HCV variations would differentially trigger host innate immunity. To this end, we stimulated human primary plasmacytoid dendritic cells (pDC) with crude preparations of different cell culture-derived genotype 2a HCV variants. Parental Japanese fulminant hepatitis C virus (JFH1) did not induce interferon alpha (IFN-α), whereas the intragenotypic chimera Jc1 triggered massive IFN-α responses. Purified Jc1 retained full infectivity but no longer induced IFN-α. Coculture of pDC with HCV-infected hepatoma cells retrieved the capacity to induce IFN-α, whereas Jc1-infected cells triggered stronger responses than JFH1-infected cells. Since the infectivity of virus particles did not seem to affect pDC activation, we next tested Jc1 mutants that were arrested at different stages of particle assembly. These experiments revealed that efficient assembly and core protein envelopment were critically needed to trigger IFN-α. Of note, sequences within domain 2 of the core that vitally affect virus assembly also crucially influenced the IFN-α responses of pDC. These data showed that viral determinants shaped host innate IFN-α responses to HCV. Although pegylated IFN-α plus ribavirin currently is the standard of care for the treatment of chronic hepatitis C virus infection, not much is known about the relevance of early interferon responses in the pathogenesis of hepatitis C virus infection. Here, we addressed whether intragenotypic variations of hepatitis C virus would account for differential induction of type I interferon responses mounted by primary blood-derived plasmacytoid dendritic cells. Surprisingly, a chimeric genotype 2a virus carrying the nonstructural genes of Japanese fulminant hepatitis C virus (JFH1) induced massive type I interferon responses, whereas the original genotype 2a JFH1 strain did not. Our detailed analyses revealed that, not the virus infectivity, but rather, the efficiency of virus assembly and core protein envelopment critically determined the magnitude of interferon responses. To our knowledge, this is the first example of hepatitis C virus-associated genetic variations that determine the magnitude of innate host responses.

Covering the Cover

Covering the Cover

HCV Activates Protein Kinase R and Attenuates Interferon-Induced MHC Class I Expression to Circumvent CD8+ T-Cell Responses

Background and AimsMajor histocompatibility complex (MHC) class I-restricted CD8+ T cells are required for clearance of hepatitis C virus (HCV) infection. MHC class I expression is up-regulated by type I and II interferons (IFNs). This study aimed to investigate the effect of HCV infection on IFN-induced expression of MHC class I and its regulatory mechanisms.MethodsHCV cell culture system (HCVcc)-infected Huh-7.5 cells with the genotype 2a Japanese fulminant hepatitis-1 strain were analyzed by flow cytometry, metabolic labeling, immunoprecipitation, and immunoblotting analyses. Protein kinase R (PKR) was knocked down with lentiviruses expressing small hairpin RNAs. Functional effects of MHC class I regulation by HCV were demonstrated in co-culture studies, using HCV-specific CD8+ T cells.ResultsBaseline level of MHC class I was not affected by HCV infection. However, IFN-induced expression of MHC class I was notably attenuated in HCV-infected cells. This was associated with replicating HCV RNA, not viral protein. HCV infection reduced IFN-induced synthesis of MHC class I protein and induced phosphorylation of PKR and eIF2α. IFN-induced MHC class I expression was restored by small hairpin RNA-mediated knockdown of PKR in HCV-infected cells. Co-culture of HCV-specific CD8+ T cells and HCV-infected cells expressing HLA-A2 demonstrated that HCV infection reduced the effector functions of HCV-specific CD8+ T cells; these were restored by small hairpin RNA-mediated knockdown of PKR.ConclusionsIFN-induced expression of MHC class I is attenuated in HCV-infected cells by activation of PKR, which reduces the effector functions of HCV-specific CD8+ T cells. This appears to be an important mechanism by which HCV circumvents antiviral adaptive immune responses. Background and AimsMajor histocompatibility complex (MHC) class I-restricted CD8+ T cells are required for clearance of hepatitis C virus (HCV) infection. MHC class I expression is up-regulated by type I and II interferons (IFNs). This study aimed to investigate the effect of HCV infection on IFN-induced expression of MHC class I and its regulatory mechanisms. Major histocompatibility complex (MHC) class I-restricted CD8+ T cells are required for clearance of hepatitis C virus (HCV) infection. MHC class I expression is up-regulated by type I and II interferons (IFNs). This study aimed to investigate the effect of HCV infection on IFN-induced expression of MHC class I and its regulatory mechanisms. MethodsHCV cell culture system (HCVcc)-infected Huh-7.5 cells with the genotype 2a Japanese fulminant hepatitis-1 strain were analyzed by flow cytometry, metabolic labeling, immunoprecipitation, and immunoblotting analyses. Protein kinase R (PKR) was knocked down with lentiviruses expressing small hairpin RNAs. Functional effects of MHC class I regulation by HCV were demonstrated in co-culture studies, using HCV-specific CD8+ T cells. HCV cell culture system (HCVcc)-infected Huh-7.5 cells with the genotype 2a Japanese fulminant hepatitis-1 strain were analyzed by flow cytometry, metabolic labeling, immunoprecipitation, and immunoblotting analyses. Protein kinase R (PKR) was knocked down with lentiviruses expressing small hairpin RNAs. Functional effects of MHC class I regulation by HCV were demonstrated in co-culture studies, using HCV-specific CD8+ T cells. ResultsBaseline level of MHC class I was not affected by HCV infection. However, IFN-induced expression of MHC class I was notably attenuated in HCV-infected cells. This was associated with replicating HCV RNA, not viral protein. HCV infection reduced IFN-induced synthesis of MHC class I protein and induced phosphorylation of PKR and eIF2α. IFN-induced MHC class I expression was restored by small hairpin RNA-mediated knockdown of PKR in HCV-infected cells. Co-culture of HCV-specific CD8+ T cells and HCV-infected cells expressing HLA-A2 demonstrated that HCV infection reduced the effector functions of HCV-specific CD8+ T cells; these were restored by small hairpin RNA-mediated knockdown of PKR. Baseline level of MHC class I was not affected by HCV infection. However, IFN-induced expression of MHC class I was notably attenuated in HCV-infected cells. This was associated with replicating HCV RNA, not viral protein. HCV infection reduced IFN-induced synthesis of MHC class I protein and induced phosphorylation of PKR and eIF2α. IFN-induced MHC class I expression was restored by small hairpin RNA-mediated knockdown of PKR in HCV-infected cells. Co-culture of HCV-specific CD8+ T cells and HCV-infected cells expressing HLA-A2 demonstrated that HCV infection reduced the effector functions of HCV-specific CD8+ T cells; these were restored by small hairpin RNA-mediated knockdown of PKR. ConclusionsIFN-induced expression of MHC class I is attenuated in HCV-infected cells by activation of PKR, which reduces the effector functions of HCV-specific CD8+ T cells. This appears to be an important mechanism by which HCV circumvents antiviral adaptive immune responses. IFN-induced expression of MHC class I is attenuated in HCV-infected cells by activation of PKR, which reduces the effector functions of HCV-specific CD8+ T cells. This appears to be an important mechanism by which HCV circumvents antiviral adaptive immune responses.

Hepatitis C Virus Infection Induces Autocrine Interferon Signaling by Human Liver Endothelial Cells and Release of Exosomes, Which Inhibits Viral Replication

Liver sinusoidal endothelial cells (LSECs) make up a large proportion of the nonparenchymal cells in the liver. LSECs are involved in induction of immune tolerance, but little is known about their functions during hepatitis C virus (HCV) infection. Primary human LSECs (HLSECs) and immortalized liver endothelial cells (TMNK-1) were exposed to various forms of HCV, including full-length transmitted/founder virus, sucrose-purified Japanese fulminant hepatitis-1 (JFH-1), a virus encoding a luciferase reporter, and the HCV-specific pathogen-associated molecular pattern molecules. Cells were analyzed by confocal immunofluorescence, immunohistochemical, and polymerase chain reaction assays. HLSECs internalized HCV, independent of cell-cell contacts; HCV RNA was translated but not replicated. Through pattern recognition receptors (Toll-like receptor 7 and retinoic acid-inducible gene 1), HCV RNA induced consistent and broad transcription of multiple interferons (IFNs); supernatants from primary HLSECs transfected with HCV-specific pathogen-associated molecular pattern molecules increased induction of IFNs and IFN-stimulated genes in HLSECs. Recombinant type I and type III IFNs strongly up-regulated HLSEC transcription of IFN λ3 (IFNL3) and viperin (RSAD2), which inhibit replication of HCV. Compared with CD8(+) T cells, HLSECs suppressed HCV replication within Huh7.5.1 cells, also inducing IFN-stimulated genes in co-culture. Conditioned media from IFN-stimulated HLSECs induced expression of antiviral genes by uninfected primary human hepatocytes. Exosomes, derived from HLSECs after stimulation with either type I or type III IFNs, controlled HCV replication in a dose-dependent manner. Cultured HLSECs produce factors that mediate immunity against HCV. HLSECs induce self-amplifying IFN-mediated responses and release of exosomes with antiviral activity.

Incorporation of primary patient-derived glycoproteins into authentic infectious hepatitis C virus particles.

The Japanese fulminant hepatitis-1 (JFH1)-based hepatitis C virus (HCV) infection system has permitted analysis of the complete viral replication cycle in vitro. However, lack of robust infection systems for primary, patient-derived isolates limits systematic functional studies of viral intrahost variation and vaccine development. Therefore, we aimed at developing cell culture models for incorporation of primary patient-derived glycoproteins into infectious HCV particles for in-depth mechanistic studies of envelope gene function. To this end, we first constructed a packaging cell line expressing core, p7, and NS2 based on the highly infectious Jc1 genotype (GT) 2a chimeric genome. We show that this packaging cell line can be transfected with HCV replicons encoding cognate Jc1-derived glycoprotein genes for production of single-round infectious particles by way of trans-complementation. Testing replicons expressing representative envelope protein genes from all major HCV genotypes, we observed that virus production occurred in a genotype- and isolate-dependent fashion. Importantly, primary GT 2 patient-derived glycoproteins were efficiently incorporated into infectious particles. Moreover, replacement of J6 (GT 2a) core, p7, and NS2 with GT 1a-derived H77 proteins allowed production of infectious HCV particles with GT 1 patient-derived glycoproteins. Notably, adaptive mutations known to enhance virus production from GT 1a-2a chimeric genomes further increased virus release. Finally, virus particles with primary patient-derived E1-E2 proteins possessed biophysical properties comparable to Jc1 HCVcc particles, used CD81 for cell entry, were associated with ApoE and could be neutralized by immune sera. This work describes cell culture systems for production of infectious HCV particles with primary envelope protein genes from GT 1 and GT 2-infected patients, thus opening up new opportunities to dissect envelope gene function in an individualized fashion.

Scalable spheroid model of human hepatocytes for hepatitis C infection and replication.

Developing effective new drugs against hepatitis C (HCV) virus has been challenging due to the lack of appropriate small animal and in vitro models recapitulating the entire life cycle of the virus. Current in vitro models fail to recapitulate the complexity of human liver physiology. Here we present a method to study HCV infection and replication on spheroid cultures of Huh 7.5 cells and primary human hepatocytes. Spheroid cultures are constructed using a galactosylated cellulosic sponge with homogeneous macroporosity, enabling the formation and maintenance of uniformly sized spheroids. This facilitates easy handling of the tissue-engineered constructs and overcomes limitations inherent of traditional spheroid cultures. Spheroids formed in the galactosylated cellulosic sponge show enhanced hepatic functions in Huh 7.5 cells and maintain liver-specific functions of primary human hepatocytes for 2 weeks in culture. Establishment of apical and basolateral polarity along with the expression and localization of all HCV specific entry proteins allow for a 9-fold increase in viral entry in spheroid cultures over conventional monolayer cultures. Huh 7.5 cells cultured in the galactosylated cellulosic sponge also support replication of the HCV clone, JFH (Japanese fulminant hepatitis)-1 at higher levels than in monolayer cultures. The advantages of our system in maintaining liver-specific functions and allowing HCV infection together with its ease of handling make it suitable for the study of HCV biology in basic research and pharmaceutical R&D.

Su1022 The Association of the CXCL10, Dpp-4 Gene Polymorphisms and Complementary Role for Unfavorable IL-28B Genotype in Prediction of Treatment Response in Asian Patients With Chronic Hepatitis C Virus Infection

subcloned into pSilencer 4.1-CMV puro vectors, and confirmed by sequencing. Huh7.5 cells were transfected with each plasmid before or after exposure to Japanese Fulminant Hepatitis Virus-1 (JFH-1), an HCV genotype 2a strain, capable of infecting human liver cells. Media were sampled at several time points, and HCV RNA was quantified by real time RT-qPCR. HCV RNA levels were statistically normalized to HCV RNA levels in the media used to infect cells. RESULTS: At 72 h, JFH-1 HCV RNA levels in media from Huh7.5 cells in the control (no plasmid transfection) and those transfected by HB (negative control) were similar. JFH-1 HCV RNA levels in those transfected by analogs, X-94, X-12, 5B-74 and 5B46 before exposure to JFH-1 HCV were 6.5%, 1.8%, 5.8%, and 6.1%, respectively of the HB controls (Fig. 1), (p<0.001). JFH-1 RNA levels in media from Huh7.5 cells transfected with analogs, X-94, X-12, 5B-74 and 5B-46 after exposure to JFH-1 HCV were 1.8%, 1.8%, 2.7%, and 3.7%, respectively of the HB controls (Fig. 2), (p <0.001). Furthermore, the X12 analog whose secondary structure is identical, but sequence is only 12% identical to JFH-1 was also highly effective suggesting that secondary structure rather than antisense effects are involved. CONCLUSIONS: Transfection of liver cells with HCV structural analogs before or after exposure to infectious virus can inhibit HCV replication and secretion of virus by more than 90%. Stable expression of these analogs might provide protection against HCV infection and treatment of HCV infection resulting in a survival advantage.

Role of Hepatitis C Virus Induced Osteopontin in Epithelial to Mesenchymal Transition, Migration and Invasion of Hepatocytes

Osteopontin (OPN) is a secreted phosphoprotein which has been linked to tumor progression and metastasis in a variety of cancers including hepatocellular carcinoma (HCC). Previous studies have shown that OPN is upregulated during liver injury and inflammation. However, the role of OPN in hepatitis C virus (HCV)-induced liver disease pathogenesis is not known. In this study, we determined the induction of OPN, and then investigated the effect of secreted forms of OPN in epithelial to mesenchymal transition (EMT), migration and invasion of hepatocytes. We show the induction of OPN mRNA and protein expression by HCV-infection. Our results also demonstrate the processing of precursor OPN (75 kDa) into 55 kDa, 42 kDa and 36 kDa forms of OPN in HCV-infected cells. Furthermore, we show the binding of secreted OPN to integrin αVβ3 and CD44 at the cell surface, leading to the activation of downstream cellular kinases such as focal adhesion kinase (FAK), Src, and Akt. Importantly, our results show the reduced expression of epithelial marker (E-cadherin) and induction of mesenchymal marker (N-cadherin) in HCV-infected cells. We also show the migration and invasion of HCV-infected cells using wound healing assay and matrigel coated Boyden chamber. In addition, we demonstrate the activation of above EMT markers, and the critical players involved in OPN-mediated cell signaling cascade using primary human hepatocytes infected with Japanese fulminant hepatitis (JFH)-1 HCV. Taken together, these studies suggest a potential role of OPN in inducing chronic liver disease and HCC associated with chronic HCV infection.

Hepatitis C Virus Attenuates Interferon-Induced Major Histocompatibility Complex Class I Expression and Decreases CD8+ T Cell Effector Functions

Major histocompatibility complex (MHC) class I-restricted CD8(+) T cells are required for clearance of hepatitis C virus (HCV) infection. MHC class I expression is up-regulated by type I and II interferons (IFNs). However, little is known about the effects of HCV infection on IFN-induced expression of MHC class I. We used the HCV cell culture system (HCVcc) with the genotype 2a Japanese fulminant hepatitis-1 strain to investigate IFN-induced expression of MHC class I and its regulatory mechanisms. HCVcc-infected Huh-7.5 cells were analyzed by flow cytometry, metabolic labeling, immunoprecipitation, and immunoblotting analyses. Protein kinase R (PKR) was knocked down with lentiviruses that express small hairpin RNAs. The functional effects of MHC class I regulation by HCV were demonstrated in co-culture studies, using HCV-specific CD8(+) T cells. Although the baseline level of MHC class I was not affected by HCV infection, IFN-induced expression of MHC class I was notably attenuated in HCV-infected cells. This was associated with replicating HCV RNA, not with viral protein. HCV infection reduced IFN-induced synthesis of MHC class I protein and induced phosphorylation of PKR and eIF2α. IFN-induced MHC class I expression was restored by small hairpin RNA-mediated knockdown of PKR in HCV-infected cells. Co-culture of HCV-specific CD8(+) T cells and HCV-infected cells that expressed HLA-A2 demonstrated that HCV infection reduced the effector functions of HCV-specific CD8(+) T cells; these functions were restored by small hairpin RNA-mediated knockdown of PKR. IFN-induced expression of MHC class I is attenuated in HCV-infected cells by activation of PKR, which reduces the effector functions of HCV-specific CD8(+) T cells. This appears to be an important mechanism by which HCV circumvents antiviral adaptive immune responses.

Human serum leads to differentiation of human hepatoma cells, restoration of very-low-density lipoprotein secretion, and a 1000-fold increase in HCV Japanese fulminant hepatitis type 1 titers

In this study, we differentiated the human hepatoma cell line Huh7.5 by supplementing tissue culture media with human serum (HS) and examined the production of hepatitis C virus (HCV) by these cells. We compared the standard tissue culture protocol, using media supplemented with 10% fetal bovine serum (FBS), to media supplemented with 2% HS. Cells cultured in HS undergo rapid growth arrest, have a hepatocyte-like morphology, and increase the expression of hepatocyte differentiation markers. In addition, expression of cell adhesion proteins claudin-1, occludin, and e-cadherin are also increased. The lipid droplet content of these cells is highly increased, as are key lipid metabolism regulators liver X receptor alpha, peroxisome proliferator-activated receptor (PPAR)-α, and PPAR-γ. Very-low-density lipoprotein secretion, which is absent in FBS-grown cells, is restored in Huh7.5 cells that are cultured in HS. All these factors have been implicated in the life cycle of HCV. We show that viral production of Japanese fulminant hepatitis type 1 increases 1,000-fold when cells are grown in HS, compared to standard FBS culture conditions. The virus produced under these conditions is associated with apolipoprotein B, has a lower density, higher specific infectivity, and has a longer half-life than virus produced in media supplemented with FBS. We describe a convenient, cost-effective method to produce hepatocyte-like cells, which produce large amounts of virus that more closely resemble HCV present in serum of infected patients.

Hepatitis C virus impairs TLR3 signaling and inhibits IFN-λ 1 expression in human hepatoma cell line

Toll-like receptor 3 (TLR3) activation plays an important role in the innate immune responses to viral infections. We show here that the activation of TLR3 signaling pathway by poly I:C, a synthetic mimic of dsRNA, could induce high-level expression of interferon (IFN)-λ1 in a hepatoma cell line. The induced IFN-λ1 contributed to poly I:C-mediated inhibition of hepatitis C virus (HCV) Japanese fulminant hepatitis-1 (JFH-1) replication in Huh7 cells. This inhibitory effect of poly I:C on HCV replication, however, was compromised by HCV infection of Huh7 cells. Investigation of the mechanisms showed that HCV infection suppressed the expression of poly I:C-induced IFN-λ1 and IFN-stimulated genes [IFN-stimulated gene 56 (ISG-56), myxovirus resistance A (MxA) and 2'-5'-oligoadenylate synthetase 1 (OAS-1))], the key antiviral elements in IFN signaling pathway. Among the HCV nonstructural (NS) proteins tested, NS3/4A, NS5A and NS5B had the ability to inhibit poly I:C-induced IFN-λ1 expression in Huh7 cells. These observations provide the experimental evidence that HCV and its proteins impair TLR3 signaling and inhibit intracellular IFN-λ1/ISG expression in a hepatoma cell line, which may account for HCV persistence in the liver.

Cd81/Cd9 Tetraspanins Aid Plasmacytoid Dendritic Cells in Recognition of Hepatitis C Virus–Infected Cells And Induction of Interferon-Alpha

Recognition of hepatitis C virus (HCV)-infected hepatocyes and interferon (IFN) induction are critical in antiviral immune response. We hypothesized that cell-cell contact between plasmacytoid dendritic cells (pDCs) and HCV-infected cells was required for IFN-α induction through the involvement of cell-surface molecules. Coculture of human peripheral blood mononuclear cells (PBMCs) with genotype 1a full-length (FL) HCV genomic replicon cells or genotype 2a Japanese fulminant hepatitis type 1 (JFH-1) virus-infected hepatoma cells (JFH-1), and not with uninfected hepatoma cells (Huh7.5), induced IFN-α production. Depletion of pDCs from PBMCs attenuated IFN-α release, and purified pDCs produced high levels of IFN-α after coculture with FL replicons or JFH-1-infected cells. IFN-α induction by HCV-containing hepatoma cells required viral replication, direct cell-cell contact with pDCs, and receptor-mediated endocytosis. We determined that the tetraspanin proteins, CD81 and CD9, and not other HCV entry receptors, were required for IFN-α induction in pDCs by HCV-infected hepatoma cells. Disruption of cholesterol-rich membrane microdomains, the localization site of CD81, or inhibition of the CD81 downstream molecule, Rac GTPase, inhibited IFN-α production. IFN-α induction involved HCV RNA and Toll-like receptor (TLR) 7. IFN-α production by HCV-infected hepatoma cells was decreased in pDCs from HCV-infected patients, compared to healthy controls. We found that preexposure of healthy PBMCs to HCV viral particles attenuated IFN-α induction by HCV-infected hepatoma cells or TLR ligands, and this inhibitory effect could be prevented by an anti-HCV envelope glycoprotein 2-blocking antibody. Our novel data show that recognition of HCV-infected hepatoma cells by pDCs involves CD81- and CD9-associated membrane microdomains and induces potent IFN-α production.