glycoprotein-e2--hepatitis-c-virus has been researched along with Liver-Neoplasms* in 22 studies
1 trial(s) available for glycoprotein-e2--hepatitis-c-virus and Liver-Neoplasms
21 other study(ies) available for glycoprotein-e2--hepatitis-c-virus and Liver-Neoplasms
| Article | Year |
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Escape of Hepatitis C Virus from Epitope I Neutralization Increases Sensitivity of Other Neutralization Epitopes.
The hepatitis C virus (HCV) E2 glycoprotein is a major target of the neutralizing antibody (nAb) response, with multiple type-specific and broadly neutralizing antibody (bnAb) epitopes identified. The 412-to-423 region can generate bnAbs that block interaction with the cell surface receptor CD81, with activity toward multiple HCV genotypes. In this study, we reveal the structure of rodent monoclonal antibody 24 (MAb24) with an extensive contact area toward a peptide spanning the 412-to-423 region. The crystal structure of the MAb24-peptide 412-to-423 complex reveals the paratope bound to a peptide hairpin highly similar to that observed with human MAb HCV1 and rodent MAb AP33, but with a different angle of approach. In viral outgrowth experiments, we demonstrated three distinct genotype 2a viral populations that acquired resistance to MAb24 via N415D, N417S, and N415D/H386R mutations. Importantly, the MAb24-resistant viruses exhibited significant increases in sensitivity to the majority of bnAbs directed to epitopes within the 412-to-423 region and in additional antigenic determinants located within E2 and the E1E2 complex. This study suggests that modification of N415 causes a global change in glycoprotein structure that increases its vulnerability to neutralization by other antibodies. This finding suggests that in the context of an antibody response to viral infection, acquisition of escape mutations in the 412-to-423 region renders the virus more susceptible to neutralization by other specificities of nAbs, effectively reducing the immunological fitness of the virus. A vaccine for HCV that generates polyspecific humoral immunity with specificity for the 412-to-423 region and at least one other region of E2 is desirable. Topics: Antibodies, Monoclonal; Antibodies, Neutralizing; Antigen-Antibody Complex; Carcinoma, Hepatocellular; Cell Line, Tumor; Epitopes; Hepacivirus; Hepatitis C Antibodies; Humans; Liver Neoplasms; Protein Structure, Secondary; Tetraspanin 28; Viral Envelope Proteins; Viral Hepatitis Vaccines | 2018 |
A protein coevolution method uncovers critical features of the Hepatitis C Virus fusion mechanism.
Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and in vitro assays, we also uncovered functionally-significant coevolving signals between E1 and E2 BL/Stem regions that govern HCV fusion, demonstrating the accuracy of our coevolution predictions. Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the development of innovative translational strategies against challenging human-tropic viruses. Topics: Animals; Carcinoma, Hepatocellular; Evolution, Molecular; Hepacivirus; Hepatitis C; Humans; Liver Neoplasms; Mice; Mice, Inbred C57BL; Protein Binding; Tumor Cells, Cultured; Viral Envelope Proteins; Virus Internalization; Virus Replication | 2018 |
AP1S3 is required for hepatitis C virus infection by stabilizing E2 protein.
Hepatitis C virus (HCV) infects 130 million people worldwide and is a leading cause of liver cirrhosis, end-stage liver disease and hepatocellular carcinoma. The interactions between viral elements and host factors play critical role on HCV invade, replication and release. Here, we identified adaptor protein complex 1 sigma 3 subunit (AP1S3) as a dependency factor for the efficient HCV infection in hepatoma cells. AP1S3 silencing in cultivated Huh7.5.1 cells significantly reduced the production of HCV progeny particles. Immunoprecipitation analysis revealed that AP1S3 interacted with the HCV E2 protein. With this interaction, AP1S3 could protect HCV E2 from ubiquitin-mediated proteasomal degradation. Using in vivo ubiquitylation assay, we identified that E6-Associated Protein (E6AP) was associated with HCV E2. In addition, treatment with synthetic peptide that contains the AP1S3-recognized motif inhibited HCV infection in Huh7.5.1 cells. Our data reveal AP1 as a novel host network that is required by viruses during infection and provides a potential target for developing broad-spectrum anti-virus strategies. Topics: Adaptor Protein Complex sigma Subunits; Carcinoma, Hepatocellular; Cell Line, Tumor; Gene Silencing; Hepacivirus; Host-Pathogen Interactions; Humans; Immunoprecipitation; Liver Neoplasms; Peptides; Proteasome Endopeptidase Complex; RNA, Viral; Ubiquitin; Ubiquitin-Protein Ligases; Viral Envelope Proteins; Virus Assembly | 2016 |
Critical interaction between E1 and E2 glycoproteins determines binding and fusion properties of hepatitis C virus during cell entry.
Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are important mediators for productive cell entry. However, knowledge about their structure, intra- or intermolecular dialogs, and conformational changes is scarce, limiting the design of therapeutic strategies targeting E1E2. Here we sought to investigate how certain domains of E1 and E2 have coevolved to optimize their interactions to promote efficient HCV entry. For this purpose we generated chimeric E1E2 heterodimers derived from two HCV 1a strains to identify and characterize crosstalk between their domains. We found an E1E2 combination that drastically impaired the infectivity of cell culture-derived HCV particles, whereas the reciprocal E1E2 combination led to increased infectivity. Using HCV pseudoparticle assays, we confirmed the opposing entry phenotypes of these heterodimers. By mutagenesis analysis, we identified a particular crosstalk between three amino acids of E1 and the domain III of E2. Its modulation leads to either a full restoration of the functionality of the suboptimal heterodimer or a destabilization of the functional heterodimer. Interestingly, we found that this crosstalk modulates E1E2 binding to HCV entry receptors SR-BI and CD81. In addition, we found for the first time that E1E2 complexes can interact with the first extracellular loop of Claudin-1, whereas soluble E2 did not. These results highlight the critical role of E1 in the modulation of HCV binding to receptors. Finally, we demonstrated that this crosstalk is involved in membrane fusion.. These results reveal a multifunctional and crucial interaction between E1 and E2 for HCV entry into cells. Our study highlights the role of E1 as a modulator of HCV binding to receptors and membrane fusion, underlining its potential as an antiviral target. Topics: Amino Acid Sequence; Animals; Carcinoma, Hepatocellular; Claudin-1; Dimerization; HEK293 Cells; Hepacivirus; Hepatitis C; Humans; Liver Neoplasms; Membrane Fusion; Molecular Sequence Data; Protein Binding; Protein Structure, Tertiary; Rats; Scavenger Receptors, Class B; Tetraspanin 28; Viral Envelope Proteins | 2014 |
HCV glycoprotein E2 is a novel BDCA-2 ligand and acts as an inhibitor of IFN production by plasmacytoid dendritic cells.
The elimination of hepatitis C virus (HCV) in > 50% of chronically infected patients by treatment with IFN-α suggests that plasmacytoid dendritic cells (pDCs), major producers of IFN-α, play an important role in the control of HCV infection. However, despite large amounts of Toll-like receptor 7-mediated IFN-α, produced by pDCs exposed to HCV-infected hepatocytes, HCV still replicates in infected liver. Here we show that HCV envelope glycoprotein E2 is a novel ligand of pDC C-type lectin immunoreceptors (CLRs), blood DC antigen 2 (BDCA-2) and DC-immunoreceptor (DCIR). HCV particles inhibit, via binding of E2 glycoprotein to CLRs, production of IFN-α and IFN-λ in pDCs exposed to HCV-infected hepatocytes, and induce in pDCs a rapid phosphorylation of Akt and Erk1/2, in a manner similar to the crosslinking of BDCA-2 or DCIR. Blocking of BDCA-2 and DCIR with Fab fragments of monoclonal antibodies preserves the capacity of pDCs to produce type I and III IFNs in the presence of HCV particles. Thus, negative interference of CLR signaling triggered by cell-free HCV particles with Toll-like receptor signaling triggered by cell-associated HCV results in the inhibition of the principal pDC function, production of IFN. Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cells, Cultured; Chlorocebus aethiops; COS Cells; Dendritic Cells; Extracellular Signal-Regulated MAP Kinases; Flow Cytometry; Hepacivirus; Host-Pathogen Interactions; Humans; Interferons; Lectins, C-Type; Ligands; Liver Neoplasms; Membrane Glycoproteins; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-akt; Receptors, Immunologic; Toll-Like Receptor 7; Viral Envelope Proteins | 2012 |
Identification of ionotrophic purinergic receptors in Huh-7 cells and their response towards structural proteins of HCV genotype 3a.
Hepatitis C virus (HCV) is a major health problem in developing countries including Pakistan. Chronic HCV infection results in progressive liver disease including fibrosis, cirrhosis, insulin resistance and eventually hepatocellular carcinoma (HCC). Ionotrophic purinergic (P2X) receptors are identified to involve in a spectrum of physiological and pathophysiological processes. However, the role of P2X receptors in HCV liver associated diseases still remains to be investigated. The current study was designed to identify the presence of P2X receptors in human liver cells. Furthermore, it investigates the response of P2X receptors towards HCV structural proteins (E1E2). To determine that how many isoforms of P2X receptors are expressed in human liver cells, human hepatoma cell line (Huh-7) was used. Transcripts (mRNA) of five different isoforms of P2X receptors were identified in Huh-7 cells. To examine the gene expression of identified isoforms of P2X receptors in presence of HCV structural proteins E1E2, Huh-7/E1E2 cell line (stably expressing HCV structural proteins E1E2) was used. The results showed significant increase (6.2 fold) in gene expression of P2X4 receptors in Huh-7/E1E2 cells as compared to control Huh-7 cells. The findings of present study confirmed the presence of transcripts of five different isoforms of P2X receptors in human liver cells and suggest that P2X4 receptors could be represented an important component of the purinergic signaling complex in HCV induced liver pathogenesis. Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Gene Expression; Genotype; Hepacivirus; Hepatitis C; Humans; Liver; Liver Cirrhosis; Liver Neoplasms; Plasmids; Protein Isoforms; Real-Time Polymerase Chain Reaction; Receptors, Purinergic P2X4; RNA, Messenger; Signal Transduction; Transfection; Viral Envelope Proteins | 2011 |
Low pH-dependent hepatitis C virus membrane fusion depends on E2 integrity, target lipid composition, and density of virus particles.
Hepatitis C virus (HCV) is an enveloped, positive strand RNA virus of about 9.6 kb. Like all enveloped viruses, the HCV membrane fuses with the host cell membrane during the entry process and thereby releases the genome into the cytoplasm, initiating the viral replication cycle. To investigate the features of HCV membrane fusion, we developed an in vitro fusion assay using cell culture-produced HCV and fluorescently labeled liposomes. With this model we could show that HCV-mediated fusion can be triggered in a receptor-independent but pH-dependent manner and that fusion of the HCV particles with liposomes is dependent on the viral dose and on the lipid composition of the target membranes. In addition CBH-5, an HCV E2-specific antibody, inhibited fusion in a dose-dependent manner. Interestingly, point mutations in E2, known to abrogate HCV glycoprotein-mediated fusion in a cell-based assay, altered or even abolished fusion in the liposome-based assay. When assaying the fusion properties of HCV particles with different buoyant density, we noted higher fusogenicity of particles with lower density. This could be attributable to inherently different properties of low density particles, to association of these particles with factors stimulating fusion, or to co-flotation of factors enhancing fusion activity in trans. Taken together, these data show the important role of lipids of both the viral and target membranes in HCV-mediated fusion, point to a crucial role played by the E2 glycoprotein in the process of HCV fusion, and reveal an important behavior of HCV of different densities with regard to fusion. Topics: Carcinoma, Hepatocellular; Cholesterol; Electroporation; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Hydrogen-Ion Concentration; Immunoenzyme Techniques; Immunoprecipitation; Indoles; Liposomes; Liver Neoplasms; Luciferases; RNA, Viral; Sphingomyelins; Transcription, Genetic; Tumor Cells, Cultured; Viral Envelope Proteins; Virion; Virus Internalization | 2009 |
Hepatitis C virus envelope components alter localization of hepatocyte tight junction-associated proteins and promote occludin retention in the endoplasmic reticulum.
Hepatocyte tight junctions (TJ) play key roles in characteristic liver functions, including bile formation and secretion. Infection by hepatitis C virus (HCV) may cause alterations of the liver architecture and disruption of the bile duct, which ultimately can lead to cholestasis. Herein, we employed the HCV replicon system to analyze the effect of HCV on TJ organization. TJ-associated proteins occludin, claudin-1, and Zonula Occludens protein-1 (ZO-1) disappeared from their normal localization at the border of adjacent cells in Huh7 clones harboring genomic but not subgenomic replicons expressing only the nonstructural proteins. Furthermore, cells containing genomic replicons showed a cytoplasmic accumulation of occludin in the endoplasmic reticulum (ER). TJ-associated function, measured as FITC-dextran paracellular permeability, of genomic replicon-containing cells, was also altered. Interestingly, clearance of the HCV replicon by interferon-alpha (IFN-alpha) treatment and by short hairpin RNA (shRNA) significantly restored the localization of TJ-associated proteins. Transient expression of all HCV structural proteins, but not core protein alone, altered the localization of TJ-associated proteins in Huh7 cells and in clones with subgenomic replicons. Confocal analysis showed that accumulation of occludin in the ER partially co-localized with HCV envelope glycoprotein E2. E2/occludin association was further confirmed by co-immunoprecipitation and pull-down assays. Additionally, using a cell culture model of HCV infection, we observed the cytoplasmic dot-like accumulation of occludin in infected Huh7 cells.. We propose that HCV structural proteins, most likely those of the viral envelope, promote alterations of TJ-associated proteins, which may provide new insights for HCV-related pathogenesis. Topics: Antiviral Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Claudin-1; Endoplasmic Reticulum; Gene Silencing; Genome, Viral; Hepacivirus; Hepatocytes; Humans; Interferon-alpha; Liver Neoplasms; Membrane Proteins; Occludin; Phosphoproteins; Replicon; Tight Junctions; Viral Envelope Proteins; Zonula Occludens-1 Protein | 2008 |
Efficient hepatitis C antigen immunohistological staining in sections of normal, cirrhotic and tumoral liver using a new monoclonal antibody directed against serum-derived HCV E2 glycoproteins.
Detection and localization of Hepatitis C Virus (HCV) in liver tissue is useful for diagnostic purposes as well as to elucidate the mechanisms by which the virus participates in hepatocarcinogenesis. However, so far, a sensitive method for HCV detection at the cellular level is lacking. We describe here the application of a novel antibody, D4.12.9, developed against serum-derived HCV RNA-positive particles, for the detection of E2 proteins by immunohistochemistry in fixed, archived specimens including liver biopsies of HCV-infected patients and surgical specimens of hepatocellular carcinoma. We demonstrate that D4.12.9 is a powerful tool for sensitive and specific detection of HCV, independently of viral genotype. This approach has applications to diagnosis as well as exploratory pathological studies. Topics: Antibodies, Monoclonal; Antibody Specificity; Hepacivirus; Hepatitis C; Hepatitis C Antigens; Humans; Immunohistochemistry; Liver; Liver Cirrhosis; Liver Neoplasms; Reproducibility of Results; Sensitivity and Specificity; Viral Envelope Proteins | 2007 |
Transient and stable expression of the HCV envelope glycoproteins in cell lines and primary hepatocytes transduced with a recombinant baculovirus.
A recombinant baculovirus, RecBV-E, encoding the hepatitis C virus (HCV) envelope proteins, E1 and E2, controlled by the cytomegalovirus promoter was constructed. RecBVs can infect mammalian cells, but fail to express proteins or replicate because the viral DNA promoters are not recognised. The RecBV-E transduced 86% of Huh7 cells and 22% of primary marmoset hepatocytes compared with 35% and 0.4%, respectively, after DNA transfection. Several stable cell lines were generated that constitutively expressed E1/E2 in every cell. No evidence of E1/E2-related apoptosis was noted, and the doubling times of cells were similar to that of the parental cells. A proportion of the E1/E2 was expressed on the surface of the stable cells as determined by flow cytometry and was detected by a conformation-dependent monoclonal antibody. It is likely that the continued expression of E1/E2 in the stable cells resulted from integration of the RecBV DNA. Infection of Huh7 cells, in the absence of G418 selection, failed to result in expression of the foreign gene (in this case, eGFP) beyond 14-18 days. RecBVs that express HCV genes from a CMV promoter represent an effective means by which to transduce primary hepatocytes for expression and replication studies. Topics: Animals; Baculoviridae; Callithrix; Carcinoma, Hepatocellular; Cell Line, Tumor; Cells, Cultured; Chlorocebus aethiops; COS Cells; Cytomegalovirus; Gene Expression; Hepacivirus; Hepatocytes; Humans; Liver Neoplasms; Promoter Regions, Genetic; Spodoptera; Transduction, Genetic; Viral Envelope Proteins | 2007 |
Intracellular accumulation of hepatitis C virus proteins in a human hepatoma cell line.
The establishment of HCV replicon systems strongly improved the research on the replication processes but poorly advanced our knowledge on the subcellular localization of the structural glycoproteins, mainly due to their low expression. We sought to verify whether reinforcing E1E2 expression in the context of both HCV genomic and subgenomic replicon from either homologous or heterologous strains leads to formation of supramolecular structures including structural and nonstructural proteins.. Robust expression of HCV glycoproteins was achieved by stable expression of E1E2p7 from genotype 1a and 1b.. In these cells, E1 and E2 triggered the formation of dot-like structures in which they co-localized with core and the nonstructural proteins NS3 and NS5A. Confocal microscopy analyses suggested that accumulation of HCV proteins occurs in an ER-derived subcompartment. Moreover, by labeling de novo-synthesized HCV RNA, we showed that these structures constitute a site of viral RNA synthesis.. Expression in trans of HCV glycoproteins in the context of replicative viral genome or subgenome generates accumulation of structural and nonstructural viral proteins in peculiar cytoplasmic structures. The simultaneous presence of viral RNA, structural and nonstructural protein suggests that these complexes represent not only sites of HCV replication but also potential places of viral pre-budding. Topics: Base Sequence; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA, Viral; Endoplasmic Reticulum; Gene Expression; Genome, Viral; Hepacivirus; Humans; Liver Neoplasms; Multiprotein Complexes; Replicon; Subcellular Fractions; Viral Envelope Proteins; Viral Nonstructural Proteins; Viral Proteins; Virus Replication | 2007 |
Intracellular versus cell surface assembly of retroviral pseudotypes is determined by the cellular localization of the viral glycoprotein, its capacity to interact with Gag, and the expression of the Nef protein.
Retroviral Gag and Env glycoproteins (GPs) are expressed from distinct cellular areas and need to encounter to interact and assemble infectious particles. Retroviral particles may also incorporate GPs derived from other enveloped viruses via active or passive mechanisms, a process known as "pseudotyping." To further understand the mechanisms of pseudotyping, we have investigated the capacity of murine leukemia virus (MLV) or lentivirus core particles to recruit GPs derived from different virus families: the G protein of vesicular stomatitis virus (VSV-G), the hemagglutinin from an influenza virus, the E1E2 glycoproteins of hepatitis C virus (HCV-E1E2), and the retroviral Env glycoproteins of MLV and RD114 cat endogenous virus. The parameters that influenced the incorporation of viral GPs onto retroviral core particles were (i) the intrinsic cell localization properties of both viral GP and retroviral core proteins, (ii) the ability of the viral GP to interact with the retroviral core, and (iii) the expression of the lentiviral Nef protein. Whereas the hemagglutinin and VSV-G glycoproteins were recruited by MLV and lentivirus core proteins at the cell surface, the HCV and MLV GPs were most likely recruited in late endosomes. In addition, whereas these glycoproteins could be passively incorporated on either retrovirus type, the MLV GP was also actively recruited by MLV core proteins, which, through interactions with the cytoplasmic tail of the latter GP, induced its localization to late endosomal vesicles. Finally, the expression of Nef proteins specifically enhanced the incorporation of the retroviral GPs by increasing their localization in late endosomes. Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Membrane; Chlorocebus aethiops; COS Cells; Endosomes; Gene Products, env; Gene Products, gag; Gene Products, nef; Hemagglutinins; Humans; Kidney; Lentivirus; Lentivirus Infections; Leukemia Virus, Murine; Liver Neoplasms; Membrane Glycoproteins; Retroviridae Infections; Rhabdomyosarcoma; Tumor Virus Infections; Viral Envelope Proteins | 2006 |
HCV E2 may induce apoptosis of Huh-7 cells via a mitochondrial-related caspase pathway.
One unusual characteristic of HCV is to establish chronic infection and the precise mechanisms remain unclear.. Huh-7 cells were transiently transfected with E2 and subjected to MTT assay, DNA fragmentation assay, and Western blotting to see the impact of E2 protein on apoptosis.. E2 may inhibit cell proliferation by inducing apoptosis and pro-caspases 3, 8, and 9 were cleaved and activated to result in the presence of active forms in a time-dependent fashion, which suggest that E2-induced apoptosis is caspase-dependent. Furthermore, the cytosolic level of cytochrome c was increased together with a gradually down-regulated Bcl-2 and up-regulated Bax protein expression. The continuing reduction of Bid protein and the gradual increase of tBid protein also indicated that a time-dependent increased turn-over of Bid protein into tBid. Taken together, our data suggested that HCV E2 may induce apoptosis through a mitochondrial damage-mediated caspase pathway. Topics: Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Hepatocellular; Caspases; Cell Line, Tumor; Humans; Liver Neoplasms; Mitochondria; Recombinant Proteins; Signal Transduction; Transfection; Viral Envelope Proteins | 2006 |
Hepatitis C virus E2 protein promotes human hepatoma cell proliferation through the MAPK/ERK signaling pathway via cellular receptors.
Dysregulation of mitogen-activated protein kinase (MAPK) signaling pathways by various viruses has been shown to be responsible for viral pathogenicity. The molecular mechanism by which hepatitis C virus (HCV) infection caused human liver diseases has been investigated on the basis of abnormal intracellular signal events. Current data are very limited involved in transmembrane signal transduction triggered by HCV E2 protein. Here we explored regulation of the MAPK/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway by E2 expressed in Chinese hamster oval cells. In human hepatoma Huh-7 cells, E2 specifically activated the MAPK/ERK pathway including downstream transcription factor ATF-2 and greatly promoted cell proliferation. CD81 and low density lipoprotein receptor (LDLR) on the cell surface mediated binding of E2 to Huh-7 cells. The MAPK/ERK activation and cell proliferation driven by E2 were suppressed by blockage of CD81 as well as LDLR. Furthermore, pretreatment with an upstream kinase MEK1/2 inhibitor U0126 also impaired the MAPK/ERK activation and cell proliferation induced by E2. Our results suggest that the MAPK/ERK signaling pathway triggered by HCV E2 via its receptors maintains survival and growth of target cells. Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Cell Membrane; CHO Cells; Cricetinae; Extracellular Signal-Regulated MAP Kinases; Humans; Kinetics; Liver Neoplasms; MAP Kinase Signaling System; Signal Transduction; Viral Envelope Proteins | 2005 |
An interplay between hypervariable region 1 of the hepatitis C virus E2 glycoprotein, the scavenger receptor BI, and high-density lipoprotein promotes both enhancement of infection and protection against neutralizing antibodies.
Hepatitis C virus (HCV) circulates in the bloodstream in different forms, including complexes with immunoglobulins and/or lipoproteins. To address the significance of such associations, we produced or treated HCV pseudoparticles (HCVpp), a valid model of HCV cell entry and its inhibition, with naïve or patient-derived sera. We demonstrate that infection of hepatocarcinoma cells by HCVpp is increased more than 10-fold by human serum factors, of which high-density lipoprotein (HDL) is a major component. Infection enhancement requires scavenger receptor BI, a molecule known to mediate HDL uptake into cells as well as HCVpp entry, and involves conserved amino acid positions in hypervariable region 1 (HVR1) of the E2 glycoprotein. Additionally, we show that the interaction with human serum or HDL, but not with low-density lipoprotein, leads to the protection of HCVpp from neutralizing antibodies, including monoclonal antibodies and antibodies present in patient sera. Finally, the deletion or mutation of HVR1 in HCVpp abolishes infection enhancement and leads to increased sensitivity to neutralizing antibodies/sera compared to that of parental HCVpp. Altogether, these results assign to HVR1 new roles which are complementary in helping HCV to survive within its host. Besides immune escape by mutation, HRV1 can mediate the enhancement of cell entry and the protection of virions from neutralizing antibodies. By preserving a balance between these functions, HVR1 may be essential for the viral persistence of HCV. Topics: Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Complementarity Determining Regions; Hepacivirus; Hepatitis C; Hepatitis C Antibodies; Humans; Lipoproteins, HDL; Liver Neoplasms; Mutagenesis, Site-Directed; Point Mutation; Recombinant Proteins; Viral Envelope Proteins | 2005 |
Characterization of liver histopathology in a transgenic mouse model expressing genotype 1a hepatitis C virus core and envelope proteins 1 and 2.
Hepatitis C virus (HCV) is a major cause of chronic hepatitis and hepatocellular carcinoma worldwide. The purpose of this study was to determine how the HCV structural proteins affect the dynamic structural and functional properties of hepatocytes and measure the extra-hepatic manifestations induced by these viral proteins. A transgenic mouse model was established by expressing core, E1 and E2 proteins downstream of a CMV promoter. HCV RNA was detected using RT-PCR in transgenic mouse model tissues, such as liver, kidney, spleen and heart. Expression of the transgene was analysed by real-time PCR to quantify viral RNA in different tissues at different ages. Immunofluorescence analysis revealed the expression of core, E1 and E2 proteins predominantly in hepatocytes. Lower levels of protein expression were detected in spleen and kidneys. HCV RNA and viral protein expression increased in the liver with age. Histological analysis of liver cells demonstrated steatosis in transgenic mice older than 3 months, which was more progressed with age. Electron microscopy analysis revealed alterations in nuclei, mitochondria and endoplasmic reticulum. HCV structural proteins induce a severe hepatopathy in the transgenic mouse model. These mice became more prone to liver and lymphoid tumour development and hepatocellular carcinoma. In this model, the extra-hepatic effects of HCV, which included swelling of renal tubular cells, were mild. It is likely that the HCV structural proteins mediate some of the histological alterations in hepatocytes by interfering with lipid transport and liver metabolism. Topics: Age Factors; Animals; Carcinoma, Hepatocellular; Cell Nucleus; Disease Models, Animal; Endoplasmic Reticulum; Fatty Liver; Female; Hematologic Neoplasms; Hepatitis C; Hepatocytes; Kidney; Liver; Liver Neoplasms; Male; Mice; Mice, Transgenic; Mitochondria; Viral Core Proteins; Viral Envelope Proteins | 2005 |
Another putative receptor for hepatitis C virus.
Topics: Antigens, CD; Carcinoma, Hepatocellular; CD36 Antigens; Hepacivirus; Humans; Liver Neoplasms; Membrane Proteins; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; Receptors, Virus; Recombinant Proteins; Scavenger Receptors, Class B; Tetraspanin 28; Tumor Cells, Cultured; Viral Envelope Proteins | 2003 |
Hepatitis C virus and HIV envelope proteins collaboratively mediate interleukin-8 secretion through activation of p38 MAP kinase and SHP2 in hepatocytes.
Hepatitis C virus (HCV) infects approximately 40% of human immunodeficiency virus (HIV) patients, and the resulting hepatic dysfunction that occurs is the primary cause of death in patients with co-infection. We hypothesized that hepatocytes exposed to HCV and HIV proteins might be susceptible to injury via an "innocent bystander" mechanism. To assess this, we studied the effects of envelope proteins, E2 of HCV and gp120 of HIV, in model HepG2 cells. Upon co-stimulation with HCV-E2 and HIV-gp120, we observed a potent proinflammatory response with the induction of IL-8. Furthermore, our studies revealed that HCV-E2 and HIV-gp120 act collaboratively to trigger a specific set of downstream signaling pathways that include activation of p38 mitogen-activated protein (MAP) kinase and the tyrosine phosphatase, SHP2. Both specific inhibitors of p38 MAP kinase and sodium vanadate, a potent protein-tyrosine phosphatase inhibitor, blocked IL-8 production in a dose-dependent manner. The role of p38 MAP kinase and SHP2 was further defined by transiently overexpressing dominant negative mutants of these proteins into HepG2 cells. These studies revealed that overexpression of an inactive p38 MAP kinase or SHP2 mutant partially abrogated HCV-E2- and HIV-gp120-induced IL-8 production. Further studies revealed that IL-8 induction was not mediated through activation of the NF-kappa B pathway. However, HCV-E2 plus HIV-gp120 was shown to increase the DNA binding activity of AP-1. These results emphasize that expression of the proinflammatory chemokine IL-8, induced by HCV-E2 and HIV-gp120, may be mediated through p38 MAP kinase and SHP2 in an NF-kappa B-independent manner, albeit through AP-1-driven processes. Topics: Carcinoma, Hepatocellular; Cell Line; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Hepacivirus; Hepatocytes; HIV Envelope Protein gp120; Humans; Imines; Interleukin-8; Intracellular Signaling Peptides and Proteins; Kinetics; Liver Neoplasms; Microcirculation; Mitogen-Activated Protein Kinases; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatases; Quinuclidines; Tumor Cells, Cultured; Viral Envelope Proteins | 2003 |
Hepatitis C virus (HCV) E1 and E2 protein regions that specifically bind to HepG2 cells.
Identify hepatitis C virus (HCV) sequences in E1 and E2 protein binding to HepG2.. Synthetic 20-mer long, ten-residue overlapped peptides, from E1 and E2 proteins, were tested in HepG2 or Raji cell-binding assays. Affinity constants, binding site number per cell and Hill coefficients were determined by saturation assay for high activity binding peptides (HABPs). Receptors for HepG2 cell were determined by cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.. Twelve HABPs were found in HCV genotype 1a, allowing six hepatocyte-binding sequences (HBSs) to be defined: two peptide-binding regions in E1 HABPs 4913 (YQVRNSTGLYHVTNDCPNSS) and 4918 (MTPTVATRDGKLPATQLRRHY). Four hepatocyte-binding regions were defined in E2: region-I, peptide 4931 (ETHVTGGSAGHTVSGFVSLLY); region-II, 4937-4939 (HHKFNSSGCPERLASCRPLTDFDQGWGPISYANGSGPDQR); region-III, 4943-4945 (PVYCFTPSPVVVGTTDRSGAPTYSWGENDTDVFVLNNTR) and region-IV, 4949-4952 (CGAPPCVIGGAGNNTLHCPTDCFRKHPDATYSRCGSGPWITPRCLVDYPY). The underlined sequences are most relevant in the binding process. HABPs 4913 and 4938 also bind to CD81 positive Raji cells. Region-II 4938 HABPs bind to 50 and 60kDa HepG2 cell membrane surface proteins.. Six HVRs to the HepG2 were identified. Some HABPs have been previously found to be antigenic and immunogenic. HABPs, 4918 (from E1), 4938, 4949, 4950, 4951 and 4952 (from E2) have not been previously recognised. These HABPs could be relevant to HCV invasion of hepatocytes. Topics: Amino Acid Sequence; Genotype; Hepacivirus; Hepatitis C; Humans; Iodine Radioisotopes; Liver Neoplasms; Molecular Sequence Data; Protein Binding; Tumor Cells, Cultured; Viral Envelope Proteins; Viral Proteins | 2002 |
Hepatitis C virus glycoprotein complex localization in the endoplasmic reticulum involves a determinant for retention and not retrieval.
The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2). These glycoproteins interact to form a noncovalent heterodimeric complex which in the cell accumulates in endoplasmic reticulum (ER)-like structures. The transmembrane domain of E2, at least, is involved in HCV glycoprotein complex localization in this compartment. In principle, ER localization of a protein can be the consequence of actual retention in this organelle or of retrieval from the Golgi. To determine which of these two mechanisms is responsible for HCV glycoprotein complex accumulation in the ER, the precise localization of these proteins was studied by immunofluorescence, and the processing of their glycans was analyzed. Immunolocalization of HCV glycoproteins after nocodazole treatment suggested an ER retention. In addition, HCV glycoprotein glycans were not modified by Golgi enzymes, indicating that the ER localization of these proteins is not because of their retrieval from the cis Golgi. Retention of HCV glycoprotein complexes in the ER without retrieval suggests that this compartment plays an important role for the acquisition of the envelope of HCV particles. A true retention in the ER was also observed for E2 expressed in the absence of E1 or for a chimeric protein containing the ectodomain of CD4 in fusion with the transmembrane domain of E2. These data indicate that, in HCV glycoprotein complex, the transmembrane domain of E2, at least, is responsible for true retention in the ER, without recycling through the Golgi. Topics: Animals; Carcinoma, Hepatocellular; Cell Line; Dimerization; Endoplasmic Reticulum; Golgi Apparatus; Hepacivirus; Humans; Liver Neoplasms; Nocodazole; Polysaccharides; Tumor Cells, Cultured; Viral Envelope Proteins | 1998 |
Comparison of hypervariable regions (HVR1 and HVR2) in positive- and negative-stranded hepatitis C virus RNA in cancerous and non-cancerous liver tissue, peripheral blood mononuclear cells and serum from a patient with hepatocellular carcinoma.
Hepatitis C virus (HCV) infection is associated with a wide spectrum of liver diseases including cirrhosis and hepatocellular carcinoma (HCC). Although the biological relation between the virus and cirrhosis or HCC is unclear, such variable pathogenicity may be related to the genetic heterogeneity of HCV. Genetic variability of HCV was assessed by determining the nucleotide sequence corresponding to the hypervariable regions (HVR1 and HVR2) of the putative envelope protein (E2/NS1) in positive- and negative-stranded HCV RNA from the cancerous and surrounding non-cancerous liver tissue, peripheral blood mononuclear cells and serum of a patient with HCC. Nineteen distinct HVR1 amino acid sequences (deduced from the nucleotide sequences) were obtained from the patient and could be classified into 5 groups on the basis of the site and time of detection. Some viral isolates with the same HVR1 sequence were shown to replicate in both cancerous and non-cancerous liver tissue, whereas others replicated in HCC tissue only. Topics: Amino Acid Sequence; Base Sequence; Carcinoma, Hepatocellular; Hepacivirus; Hepatitis C; Humans; Liver; Liver Neoplasms; Male; Middle Aged; Molecular Sequence Data; RNA, Viral; Sequence Analysis; Viral Envelope Proteins | 1996 |