Hepatitis C Virus Core Protein Down-Regulates Expression of Src-Homology 2 Domain Containing Protein Tyrosine Phosphatase by Modulating Promoter DNA Methylation.

Hepatitis C virus (HCV) is the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma. The main virion component, the core (C) protein, has been implicated in several aspects of HCV pathology including oncogenesis and immune subversion. Here we show that expression of the C protein induced specific tyrosine phosphorylation of the TCR-related signaling proteins ZAP-70, LAT and PLC-γ in the T cells. Stable expression of the C protein specifically reduced Src homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1) mRNA and protein accumulation. Quantitative CpG methylation analysis revealed a distinct CpG methylation pattern at the SHP-1 gene promoter in the C protein expressing cells that included specific hypermethylation of the binding site for Sp1 transcription factor. Collectively, our results suggest that HCV may suppress immune responses and facilitate its own persistence by deregulating phosphotyrosine signaling via repressive epigenetic CpG modification at the SHP-1 promoter in the T cells.

An electrochemical biosensor for direct detection of hepatitis C virus.

This paper is aimed at the development of a biosensor for direct detection of Hepatitis C virus (HCV) surface antigen: envelope protein (E2). A recombinant LEL fragment of biological cell receptor CD81 and two short synthetic peptides imitating the fragment of LEL sequence of CD81 (linear and loop-like peptides) capable of specific binding to E2 were tested as molecular recognition elements of the biosensor. For this purpose the selected ligands were immobilized to the surface of a screen-printed electrode utilized as an electrochemical sensor platform. The immobilization parameters such as the ligand concentration and the immobilization time were carefully optimized for each ligand. Differential pulse voltammetry used to evaluate quantitatively binding of E2 to the ligands revealed their similar binding affinity towards E2. Thus, the linear peptide was selected as a less expensive and easily prepared ligand for the HCV biosensor preparation. The resulting HCV biosensor demonstrated selectivity towards E2 in the presence of interfering protein, conalbumin. Moreover, it was found that the prepared biosensor effectively detected E2 bound to hepatitis C virus-mimetic particles (HC VMPs) at LOD value of 2.1∙10-5 mg/mL both in 0.01 M PBS solution (pH 7.4) and in simulated blood plasma.

Optimized cell systems for the investigation of hepatitis C virus E1E2 glycoproteins.

Great strides have been made in understanding and treating hepatitis C virus (HCV) thanks to the development of various experimental systems including cell-culture-proficient HCV, the HCV pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterizing neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones produce pseudoparticles that are either non-infectious or exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate-binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293TCD81KO). Clones made in 293TCD81KO cells were antigenically very similar to their matched counterparts made parental cells and appear to honour the accepted HCV entry pathway. Deletion of CD81 did not appreciably increase the recovered titres of soluble E2 (sE2). However, we did, unexpectedly, find that monomeric sE2 made in 293T cells and Freestyle 293-F (293-F) cells exhibit important differences. We found that 293-F-produced sE2 harbours mostly complex-type glycans whilst 293T-produced sE2 displays a heterogeneous mixture of both complex-type glycans and high-mannose or hybrid-type glycans. Moreover, sE2 produced in 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293-F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens.

Cortactin Interacts with Hepatitis C Virus Core and NS5A Proteins: Implications for Virion Assembly.

Hepatitis C virus (HCV) exploits cellular proteins to facilitate viral propagation. To identify the cellular factors involved in the HCV life cycle, we previously performed protein microarray assays using either HCV nonstructural 5A (NS5A) protein or core protein as a probe. Interestingly, cellular cortactin strongly interacted with both NS5A and core. Cortactin is an actin-binding protein critically involved in tumor progression by regulating the migration and invasion of cancerous cells. Protein interaction between cortactin and NS5A or core was confirmed by coimmunoprecipitation and immunofluorescence assays. We showed that cortactin interacted with NS5A and core via the N-terminal acidic domain of cortactin. Cortactin expression levels were not altered by HCV infection. Small interfering RNA (siRNA)-mediated knockdown of cortactin dramatically decreased HCV protein expression and infectivity levels, whereas overexpression of cortactin increased viral propagation. Ectopic expression of the siRNA-resistant cortactin recovered the viral infectivity, suggesting that cortactin was specifically required for HCV propagation. We further showed that cortactin was involved in the assembly step without affecting viral entry, HCV internal ribosome entry site (IRES)-mediated translation, and the replication steps of the HCV life cycle. Of note, silencing of cortactin markedly reduced both NS5A and core protein levels on the lipid droplets (LDs), and this effect was reversed by the overexpression of cortactin. Importantly, NS5A and core promoted cell migration by activating the phosphorylation of cortactin at tyrosine residues 421 and 466. Taken together, these data suggest that cortactin is not only involved in HCV assembly but also plays an important role in the cell migration.IMPORTANCE Cortactin is a cytoskeletal protein that regulates cell migration in response to a number of extracellular stimuli. The functional involvement of cortactin in the virus life cycle is not yet fully understood. The most significant finding is that cortactin strongly interacted with both hepatitis C virus (HCV) core and NS5A. Cortactin is involved in HCV assembly by tethering core and NS5A on the lipid droplets (LDs) with no effect on LD biogenesis. It was noteworthy that HCV NS5A and core activated cortactin by phosphorylation at tyrosines 421 and 466 to regulate cell migration. Collectively, our study shows that cortactin is a novel host factor involved in viral production and HCV-associated pathogenesis.

The HBx and HBc of hepatitis B virus can influence Id1 and Id3 by reducing their transcription and stability.

Hepatitis B virus (HBV) infection is closely related with the occurrence and development of hepatocellular carcinoma (HCC), in which Hepatitis B virus x protein (HBx) and core protein (HBc) play crucial roles. Additionally, inhibitors of differentiation (Id) proteins exhibited significant correlation with liver cancer development. Here, we identified that HBV dramatically inhibited the expression of Id1 and Id3 in both protein and transcriptional levels for the first time, whereas there was little effect of the virus on Id2. Additionally, two HBV coded protein, HBc and HBx, could reduce the expression of Id1 and Id3 distinctly, whereas the other two viral proteins, HBs and HBp were unable to affect Id1 and Id3 proteins. Both the activity inhibitors and activators further confirmed that HBc inhibited the expression of Id1 and Id3 by BMP/Smad signaling pathway. HBx could interact with both Id1 and Id3 at residues 112-136 of HBx protein, and it could inhibit the two Id proteins by accelerating their degradation. This is the first report about HBc and HBx regulating Id1 and Id3, whereas the detailed mechanism associated with above needed further experiments to clarify.

Clinical utility of hepatitis C virus core antigen assay in the monitoring of direct-acting antivirals for chronic hepatitis C.

BACKGROUND: Hepatitis C virus core antigen (HCV Ag) assay has been proposed as a more economical alternative to HCV RNA detection. This study aimed to investigate the clinical utility of HCV Ag assay in the monitoring of direct-acting antivirals (DAAs) for chronic hepatitis C patients. METHODS: We analyzed serum samples from 110 patients treated with paritaprevir/ritonavir, ombitasvir, and dasabuvir (PrOD) with or without ribavirin. The levels for both HCV Ag and HCV RNA assessed by COBAS TaqMan HCV (CTM) Test or Abbott RealTime HCV (ART) assay were evaluated at baseline, week 2, 4, and 12 during treatment and 12 weeks after completion. RESULTS: Baseline HCV Ag levels showed good correlations with HCV viral load (r = 0.879; p<0.001); whereas the correlation was slightly stronger with CTM test than with ART assay (p = 0.074). The concordance of HCV Ag and HCV RNA undetectability was significantly better in CTM test than in ART assay at week 2 (p = 0.003) and week 4 (p = 0.003). A sustained viral response 12 weeks off therapy (SVR12) was achieved in 108 patients (98%); the HCV Ag assay identified 99% of these patients. Both undetectability of serum HCV Ag and HCV RNA had high positive predictive value at week 2 (98% vs. 100%) and at week 4 (97% vs. 99%) in predicting SVR12. CONCLUSIONS: HCV Ag assay may be a feasible alternative to HCV RNA for the determination of SVR12 in patients treated with DAAs.

Hepatitis C core antigen: Diagnosis and monitoring of patients infected with hepatitis C virus.

INTRODUCTION: New efficient strategies are needed for the assessment of active hepatitis C virus (HCV) infection. The aim of this study was to evaluate the ability of HCV core antigen (HCV-cAg) as a marker of active HCV infection in newly diagnosed patients, for treatment monitoring, and for the detection of therapeutic failure. MATERIALS AND METHODS: A prospective study was conducted at a regional reference hospital in Spain. HCV-cAg and viral load (RNA-HCV) were tested in plasma or serum samples from three patient groups: new diagnosis, treatment monitoring, and treatment failure. The treatment monitoring group was tested at the beginning of treatment, at 4 weeks post-initiation, at the end of treatment, and at 12 weeks post-treatment completion. The Architect HCV core antigen assay was performed for HCV-cAg testing, and viral load was quantified with the Cobas 6800 system. RESULTS: A total of 303 samples from 124 patients were analyzed. Excellent correlation was seen between HCV-cAg and HCV-RNA (R2=0.932). The optimal cut-off value was 3fmol/l in the receiver operating characteristics curve analysis, and the area under the curve was 0.987 (95% confidence interval 0.972-1.000). HCV-cAg sensitivity and specificity were 97% and 95%, respectively. Most diverging results were observed in the treatment follow-up group. CONCLUSIONS: HCV-cAg demonstrated good sensitivity and specificity as a marker for active HCV infection, new diagnosis, detection of antiviral therapeutic failure, and treatment monitoring.

Clinical utility of HCV core antigen detection and quantification using serum samples and dried blood spots in people who inject drugs in Dar-es-Salaam, Tanzania.

INTRODUCTION: A lack of access to hepatitis C virus (HCV) diagnostics is a significant barrier to achieving the World Health Organization 2030 global elimination goal. HCV core antigen (HCVcAg) quantification and dried blood spot (DBS) are appealing alternatives to conventional HCV serology and nucleic acid testing (NAT) for resource-constraint settings, particularly in difficult-to-reach populations. We assessed the accuracy of serum and DBS HCVcAg testing in people who inject drugs in Tanzania using HCV NAT as a reference. METHOD: Between May and July 2015, consecutive HCV-seropositive patients enrolled in the local opioid substitution treatment centre were invited to participate in the study. All had HCV RNA detection (Roche Molecular Systems, Pleasanton, CA, USA), genotyping (NS5B gene phylogenetic analysis) and HCVcAg on blood samples and DBS (Architect assay; Abbott Diagnostics, Chicago, IL, USA). RESULTS: Out of 153 HCV-seropositive individuals, 65 (42.5%) and 15 (9.8%) were co-infected with HIV (41 (63%) were on anti-retroviral therapy (ARVs)) and hepatitis B respectively. In total, 116 were viraemic, median viral load of 5.7 (Interquartile range (IQR); 4.0-6.3) log iU/ml (75 (68.2%) were genotype 1a, 35 (31.8%) genotype 4a). The median alanine transaminase (ALT) (iU/l), aspartate transaminase (AST) (iU/l) and gamma-glutamyl transferase (GGT) (iU/l) were 35 (IQR; 23-51), 46 (32-57) and 69 (35-151) respectively. For the quantification of HCV RNA, serum HCVcAg had a sensitivity at 99.1% and a specificity at 94.1%, with an area under the receiver operating curve (AUROC) at 0.99 (95% CI 0.98-1.00). DBS HCVcAg had a sensitivity of 76.1% and a specificity of 97.3%, with an AUROC of 0.87 (95% CI 0.83-0.92). HCVcAg performance did not differ by HIV co-infection or HCV genotype. Conclusions Our study suggests that HCVcAg testing in serum is an excellent alternative to HCV polymerase chain reaction in Africa. Although HCVcAg detection and quantification in DBS has a reduced sensitivity, its specificity and accuracy are good and it could therefore be used for scaling up HCV testing and care in resource-limited African settings.

Hepatitis C virus core antigen: A potential alternative to HCV RNA testing among persons with substance use disorders.

BACKGROUND: The hepatitis C virus (HCV) core antigen (HCVcAg) may be an alternative diagnostic method to HCV RNA especially in populations such as substance users, the homeless or in resource-limited settings. AIMS: To evaluate performance of HCVcAg test in patients with opioid use disorder (OUD) on methadone in order to document its performance characteristics in the target population and to ensure that its specificity remains consistent across different populations. METHODS: HCVcAg levels from 109 methadone-maintained patients were compared to HCV RNA levels. RESULTS: Mean age was 53.8±7.8years, 59.6% were male, 68.8% African American, and 44% HCV-infected. HCVcAg was detectable in 47 of 48 HCV-infected, and undetectable in all HCV RNA negative patients. The HCVcAg assay had sensitivity of 97.9% and specificity of 100%. Correlation with HCV RNA levels was excellent (r=0.88, 95% CI 0.76; 0.95, p<0.01). CONCLUSION: HCVcAg has excellent performance for the diagnosis of HCV infection in patients with OUD on methadone.

Frequent detection of HCV RNA and HCVcoreAg in stool of patients with chronic hepatitis C.

BACKGROUND AND OBJECTIVE: HCV is transmitted mainly by parenteral routes. However, unprotected anal intercourse has also been identified as a risk factor for HCV infection. HCV RNA can be detected in blood, saliva, and bile, but the presence of HCV in stool has not been investigated yet. STUDY DESIGN: Therefore, stool samples of 98 patients were collected prospectively. Specific HCV primers were used to identify samples positive for HCV RNA. HCV RNA-positive samples were tested for HCVcoreAg with the Architect HCVAg assay (Abbott). Presence of occult blood was investigated by the hemoCARE guajak test. Viral stability and infectivity of recombinant HCV particles was investigated in vitro by incubation of genotype 2a chimeric virus Jc1 with bile and stool suspensions. RESULTS: HCV RNA could be detected in 68 out of 98 stool samples from patients with chronic hepatitis C and 16 samples also tested positive for HCVcoreAg. Presence of HCV RNA in stool was more frequent in male than in female and in patients with low platelet counts but was not associated with the detection of occult blood. Stool suspensions and to a lesser extent bile reduced the in vitro infectivity of genotype 2a chimeric Jc1 virus even though infection of Huh7 cells was not completely abrogated. CONCLUSIONS: In summary, this study shows for the first time that HCV can frequently be detected in stool samples of chronically infected patients irrespective of occult bleeding. We suggest that stool can be a potential source for HCV infection and thus unprotected anal intercourse should be avoided.

Hepatitis C virus core protein modulates pRb2/p130 expression in human hepatocellular carcinoma cell lines through promoter methylation.

BACKGROUND: Hepatitis C Virus (HCV) infection is associated with chronically evolving disease and development of hepatocellular carcinoma (HCC), albeit the mechanism of HCC induction by HCV is still controversial. The nucleocapsid (core) protein of HCV has been shown to be directly implicated in cellular transformation and immortalization, enhancing the effect of oncogenes and decreasing the one of tumor suppressor genes, as RB1 and its protein product pRB. With the aim of identifying novel molecular mechanisms of hepatocyte transformation by HCV, we examined the effect of HCV core protein on the expression of the whole Retinoblastoma (RB) family of tumor and growth suppressor factors, i.e. pRb, p107 and pRb2/p130. METHODS: We used a model system consisting of the HuH-7, HCV-free, human hepatocellular carcinoma cell line and of the HuH-7-CORE cells derived from the former and constitutively expressing the HCV core protein. We determined pRb, p107 and pRb2/p130 protein and mRNA amount of the respective genes RB1, RBL1 and RBL2, RBL2 promoter activity and methylation as well as DNA methyltransferase 1 (DNMT1) and 3b (DNMT3b) expression level. The effect of pRb2/p130 over-expression on the HCV core-expressing HuH-7-CORE cells was also evaluated. RESULTS: We found that the HCV core protein expression down-regulated pRb2/p130 protein and mRNA levels in HuH-7-CORE cells by inducing promoter hyper-methylation with the concomitant up-regulation of DNMT1 and DNMT3b expression. When pRb2/p130 expression was artificially re-established in HuH-7-CORE cells, cell cycle analysis outlined an accumulation in the G0/G1 phase, as expected. CONCLUSIONS: HCV core appears indeed able to significantly down-regulate the expression and the function of two out of three RB family tumor and growth suppressor factors, i.e. pRb and pRb2/p130. The functional consequences at the level of cell cycle regulation, and possibly of more complex cell homeostatic processes, may represent a plausible molecular mechanism involved in liver transformation by HCV.

Detergent-resistant membrane association of NS2 and E2 during hepatitis C virus replication.

UNLABELLED: Previously, we demonstrated that the efficiency of hepatitis C virus (HCV) E2-p7 processing regulates p7-dependent NS2 localization to putative virus assembly sites near lipid droplets (LD). In this study, we have employed subcellular fractionations and membrane flotation assays to demonstrate that NS2 associates with detergent-resistant membranes (DRM) in a p7-dependent manner. However, p7 likely plays an indirect role in this process, since only the background level of p7 was detectable in the DRM fractions. Our data also suggest that the p7-NS2 precursor is not involved in NS2 recruitment to the DRM, despite its apparent targeting to this location. Deletion of NS2 specifically inhibited E2 localization to the DRM, indicating that NS2 regulates this process. Treatment of cells with methyl-ß-cyclodextrin (MßCD) significantly reduced the DRM association of Core, NS2, and E2 and reduced infectious HCV production. Since disruption of the DRM localization of NS2 and E2, either due to p7 and NS2 defects, respectively, or by MßCD treatment, inhibited infectious HCV production, these proteins' associations with the DRM likely play an important role during HCV assembly. Interestingly, we detected the HCV replication-dependent accumulation of ApoE in the DRM fractions. Taking into consideration the facts that ApoE was shown to be a major determinant for infectious HCV particle production at the postenvelopment step and that the HCV Core protein strongly associates with the DRM, recruitment of E2 and ApoE to the DRM may allow the efficient coordination of Core particle envelopment and postenvelopment events at the DRM to generate infectious HCV production. IMPORTANCE: The biochemical nature of HCV assembly sites is currently unknown. In this study, we investigated the correlation between NS2 and E2 localization to the detergent-resistant membranes (DRM) and HCV particle assembly. We determined that although NS2's DRM localization is dependent on p7, p7 was not targeted to these membranes. We then showed that NS2 regulates E2 localization to the DRM, consistent with its role in recruiting E2 to the virus assembly sites. We also showed that short-term treatment with the cholesterol-extracting agent methyl-ß-cyclodextrin (MßCD) not only disrupted the DRM localization of Core, NS2, and E2 but also specifically inhibited intracellular virus assembly without affecting HCV RNA replication. Thus, our data support the role of the DRM as a platform for particle assembly process.

Functional characterization of nuclear localization and export signals in hepatitis C virus proteins and their role in the membranous web.

The hepatitis C virus (HCV) is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS) and nuclear export signals (NES) have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC) proteins (termed nucleoporins or Nups) are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin α5 (IPOA5/kapα1), importin ß3 (IPO5/kap ß3), and exportin 1 (XPO1/CRM1) both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication.

Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure.

The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV.

Hepatitis C virus core protein suppresses mitophagy by interacting with parkin in the context of mitochondrial depolarization.

Hepatitis C virus (HCV) causes mitochondrial injury and oxidative stress, and impaired mitochondria are selectively eliminated through autophagy-dependent degradation (mitophagy). We investigated whether HCV affects mitophagy in terms of mitochondrial quality control. The effect of HCV on mitophagy was examined using HCV-Japanese fulminant hepatitis-1-infected cells and the uncoupling reagent carbonyl cyanide m-chlorophenylhydrazone as a mitophagy inducer. In addition, liver cells from transgenic mice expressing the HCV polyprotein and human hepatocyte chimeric mice were examined for mitophagy. Translocation of the E3 ubiquitin ligase Parkin to the mitochondria was impaired without a reduction of pentaerythritol tetranitrate-induced kinase 1 activity in the presence of HCV infection both in vitro and in vivo. Coimmunoprecipitation assays revealed that Parkin associated with the HCV core protein. Furthermore, a Yeast Two-Hybrid assay identified a specific interaction between the HCV core protein and an N-terminal Parkin fragment. Silencing Parkin suppressed HCV core protein expression, suggesting a functional role for the interaction between the HCV core protein and Parkin in HCV propagation. The suppressed Parkin translocation to the mitochondria inhibited mitochondrial ubiquitination, decreased the number of mitochondria sequestered in isolation membranes, and reduced autophagic degradation activity. Through a direct interaction with Parkin, the HCV core protein suppressed mitophagy by inhibiting Parkin translocation to the mitochondria. This inhibition may amplify and sustain HCV-induced mitochondrial injury.

Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure

The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV.

Imaging of hepatitis C virus infection in liver grafts after liver transplantation.

BACKGROUND & AIMS: The detection of native hepatitis C virus (HCV) antigens in liver tissue may be relevant to diagnostic purposes and to better understand the pathogenesis of HCV infection. The aim of our study was to characterize HCV antigens in liver grafts. METHODS: We selected 32 liver transplant (LT) recipients with recurrent hepatitis C. HCV core and NS5A antigens were detected in formalin-fixed, paraffin-embedded (FFPE) liver biopsies obtained immediately after graft reperfusion (negative controls), during the acute phase of HCV infection (1-6 months) and during follow-up (7-74 months) after LT. Viral antigens were assessed by immunohistochemistry and confocal microscopy. RESULTS: All reperfusion biopsies were negative for both antigens. Core protein was detected in 75% and 33% of acute phase and follow-up biopsies, respectively. HCV antigens were not detected in any of the 10 samples from patients who cleared HCV after antiviral treatment. Immunostaining was hepatocellular, with a granular cytoplasmic pattern and a wide spectrum of intensity. We found a significant association between viral load and the presence of HCV core-positive hepatocytes (p=0.004). NS5A colocalized strongly with core (66%) and adipophilin (36%), supporting the localization of core and NS5A around lipid droplets. A detailed three-dimensional analysis showed that NS5A surrounded the core and adipophilin-positive areas. CONCLUSIONS: HCV antigens can be detected in FFPE liver biopsies by immunohistochemistry. The in vivo colocalization of core and NS5A proteins around the lipid droplets supports that the latter may play a role in virus particle production, similar to what reported in vitro.

Hepatitis C virus Core protein overcomes all-trans retinoic acid-induced cell growth arrest by inhibiting retinoic acid receptor-ß2 expression via DNA methylation.

Aberrant promoter methylation of tumor suppressor genes including retinoic acid receptor-ß2 (RAR-ß2) is frequently detected in hepatitis C virus (HCV)-associated hepatocellular carcinoma; however, the mechanism and its significance are relatively unknown. Here, we showed that HCV Core induced promoter hypermethylation of RAR-ß2 to inhibit its expression via up-regulation of DNA methyltransferases 1 and 3b. Under the condition, all-trans retinoic acid (ATRA) failed to activate p16 expression and thus could not inactivate the Rb-E2F pathway. Accordingly, Core-expressing cells exhibited resistance to ATRA-induced growth inhibition. Taken together, HCV Core antagonizes ATRA, a natural anti-cancer compound, to stimulate cell growth via epigenetic down-regulation of RAR-ß2.

Expression of the full-length HCV core subgenome from HCV gentoype-1a and genotype-3a and evaluation of the antigenicity of translational products.

BACKGROUND: Hepatitis C virus (HCV) infection is a major public health problem in India. Detection of HCV and its genotypes by simple and economic assays is a prime requirement in the planning of antiviral treatments for patients infected with this virus. Although commercial assays are available for the detection of both HCV RNA and genotypes, efforts aimed at the development of simple and economical systems for these measurements are still going on. AIM: The present study was designed to clone and express the HCV CORE gene from HCV genotype-1a and genotype-3a and use the peptides to develop immunoassays for the detection of genotype-specific antibodies in sera samples. METHODS: One hundred and thirty-five serum samples from patients with liver and renal diseases were screened for HCV RNA by real-time PCR, followed by HCV genotyping in RNA-positive sera by restriction fragment length polymorphism, sequencing, and phylogenetic analysis. The HCV CORE gene was amplified from sera carrying HCV genotype-1a and genotype-3a and cloned and expressed in the pET19b vector. The translational products were used to develop a western blot assay for the detection of genotype-specific anti-HCV antibodies. RESULTS: The HCV CORE gene, from both genotypes, was cloned and expressed successfully, with production of a 26 kDa recombinant protein in either case. Using peptides in a western blot assay, 101 sera samples were tested for the anti-HCV CORE antibody. Each peptide showed a reaction with anti-HCV total antibody without showing any genotype-specific binding. This indicates that individual peptides obtained from different genotypes do not have a genotype-specific epitope to bind with antibodies. CONCLUSION: Cloning and expression of the HCV CORE gene from genotype-1a and genotype-3a was successful. However, the peptides formed did not show genotype-specific binding with anti-HCV.