scholarly journals Hepatitis C Virus-Like Particle Morphogenesis

2002 ◽  
Vol 76 (8) ◽  
pp. 4073-4079 ◽  
Author(s):  
Emmanuelle Blanchard ◽  
Denys Brand ◽  
Sylvie Trassard ◽  
Alain Goudeau ◽  
Philippe Roingeard
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mammalian Cells ◽  
Semliki Forest Virus ◽  
Virus Like Particle ◽  
Genes Encoding ◽  
Host Cell Interactions ◽  
Viral Morphogenesis ◽  
First Time

ABSTRACT Although much is known about the hepatitis C virus (HCV) genome, first cloned in 1989, little is known about HCV structure and assembly due to the lack of an efficient in vitro culture system for HCV. Using a recombinant Semliki forest virus replicon expressing genes encoding HCV structural proteins, we observed for the first time the assembly of these proteins into HCV-like particles in mammalian cells. This system opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions.

scholarly journals Hepatitis C Virus-Like Particle Budding: Role of the Core Protein and Importance of Its Asp111

2003 ◽  
Vol 77 (18) ◽  
pp. 10131-10138 ◽  
Author(s):  
Emmanuelle Blanchard ◽  
Christophe Hourioux ◽  
Denys Brand ◽  
Malika Ait-Goughoulte ◽  
Alain Moreau ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Semliki Forest Virus ◽  
Core Protein ◽  
Virus Assembly ◽  
Signal Sequence ◽  
E1 Protein ◽  
Hcv Core Protein ◽  
Virus Like Particle ◽  
Genes Encoding

ABSTRACT In the absence of a hepatitis C virus (HCV) culture system, the use of a Semliki Forest virus replicon expressing genes encoding HCV structural proteins that assemble into HCV-like particles provides an opportunity to study HCV morphogenesis. Using this system, we showed that the HCV core protein constitutes the budding apparatus of the virus and that its targeting to the endoplasmic reticulum by means of the signal sequence of E1 protein is essential for budding. In addition, the aspartic acid at position 111 in the HCV core protein sequence was found to be crucial for virus assembly, demonstrating the usefulness of this system for mapping amino acids critical to HCV morphogenesis.

scholarly journals A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria

2004 ◽  
Vol 85 (2) ◽  
pp. 451-461 ◽  
Author(s):  
Stephen D. C. Griffin ◽  
Ruth Harvey ◽  
Dean S. Clarke ◽  
Wendy S. Barclay ◽  
Mark Harris ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Ion Channel ◽  
Lipid Bilayers ◽  
Mammalian Cells ◽  
Intracellular Localization ◽  
Channel Activity ◽  
Diarrhoea Virus ◽  
Basic Loop

We previously identified the function of the hepatitis C virus (HCV) p7 protein as an ion channel in artificial lipid bilayers and demonstrated that this in vitro activity is inhibited by amantadine. Here we show that the ion channel activity of HCV p7 expressed in mammalian cells can substitute for that of influenza virus M2 in a cell-based assay. This was also the case for the p7 from the related virus, bovine viral diarrhoea virus (BVDV). Moreover, amantadine was shown to abrogate HCV p7 function in this assay at a concentration that specifically inhibits M2. Mutation of a conserved basic loop located between the two predicted trans-membrane alpha helices rendered HCV p7 non-functional as an ion channel. The intracellular localization of p7 was unaffected by this mutation and was found to overlap significantly with membranes associated with mitochondria. Demonstration of p7 ion channel activity in cellular membranes and its inhibition by amantadine affirm the protein as a target for future anti-viral chemotherapy.

scholarly journals Synthesis of New Indanyl Nucleoside Analogues and their Biological Evaluation on Hepatitis C Virus (HCV) Replicon

Molecules ◽  
2019 ◽  
Vol 24 (5) ◽  
pp. 990
Author(s):  
Matías Gómez ◽  
Emiliano Gentile ◽  
M. Martini ◽  
María Cuestas ◽  
Verónica Mathet ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Biological Evaluation ◽  
Computational Studies ◽  
Replicon Cell ◽  
Racemic Form ◽  
Synthetic Procedure ◽  
Replicon Cell Line ◽  
First Time

Here, we report a convenient synthetic procedure for the preparation of four novel indanyl carbanucleoside derivatives in the racemic form. The action of these compounds against hepatitis C virus was evaluated in vitro using the replicon cell line, Huh7.5 SG. Contrary to our expectations, all these compounds did not inhibit, but rather promoted HCV genotype 1b (HCVg1b) replication. Similar effects have been reported for morphine in the replicon cell lines, Huh7 and Huh8. Several biological experiments and computational studies were performed to elucidate the effect of these compounds on HCVg1b replication. Based on all the experiments performed, we propose that the increase in HCVg1b replication could be mediated, at least in part, by a similar mechanism to that of morphine on the enhancement of this replication. The presence of opioid receptors in Huh7.5 SG cells was indirectly determined for the first time in this work.

scholarly journals Hepatitis C virus infection of primary tupaia hepatocytes leads to selection of quasispecies variants, induction of interferon-stimulated genes and NF-κB nuclear translocation

2005 ◽  
Vol 86 (11) ◽  
pp. 3065-3074 ◽  
Author(s):  
Anunciata Guitart ◽  
José-Ignacio Riezu-Boj ◽  
Edurne Elizalde ◽  
Esther Larrea ◽  
Carmen Berasain ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Semliki Forest Virus ◽  
Nuclear Translocation ◽  
Natural Infection ◽  
Hcv Infection ◽  
Interferon Response ◽  
Cell Phenotype

Systems for in vitro culture of Hepatitis C virus (HCV) are essential tools to analyse virus–cell interactions and to investigate relevant pathophysiological aspects of HCV infection. Although the HCV replicon methodology has increased our understanding of HCV biology, this system does not reproduce the natural infection. Recently, tupaia (Tupaia belangeri chinensis) hepatocytes have been utilized for in vitro culture of HCV. In the present work, primary tupaia hepatocytes infected in vitro with HCV were used to analyse the evolution of HCV quasispecies in infected cells and the ability of the virus to influence antiviral and proinflammatory responses in cells sustaining virus replication. The results confirmed the potential of tupaia hepatocytes as a model for HCV infection, although this system is limited by rapid loss of differentiated cell phenotype in culture. These findings revealed an extraordinary plasticity of HCV quasispecies, which underwent rapid evolution to tupaia-tropic variants as early as 24 h after infection. It was also shown that HCV could activate interferon-sensitive genes, albeit modestly in comparison with other viruses such as Semliki Forest virus. Importantly, HCV activated NF-κB in primary hepatocytes and upregulated NF-κB-responsive genes including the chemokines MCP-1 and CXCL2 (MIP-2). This effect may play a role in induction of the hepatic inflammatory reaction in vivo. In summary, HCV quasispecies adapt rapidly to the specific biology of the host and HCV stimulates a blunted interferon response while inducing a proinflammatory phenotype in the infected cell.

scholarly journals Hepatitis C virus non-structural protein 3-specific cellular immune responses following single or combined immunization with DNA or recombinant Semliki Forest virus particles

2002 ◽  
Vol 83 (2) ◽  
pp. 369-381 ◽  
Author(s):  
C. Brinster ◽  
M. Chen ◽  
D. Boucreux ◽  
G. Paranhos-Baccala ◽  
P. Liljeström ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Immune Responses ◽  
Semliki Forest Virus ◽  
Cytotoxic Lymphocytes ◽  
Structural Protein ◽  
Virus Particles ◽  
Cellular Immune Responses ◽  
Cellular Immune

The capacity of recombinant Semliki Forest virus particles (rSFV) expressing the hepatitis C virus non-structural protein 3 (NS3) to induce, in comparison or in combination with an NS3-expressing plasmid, specific cellular and humoral immune responses in murine models was evaluated. In vitro studies indicated that both types of vaccine expressed the expected size protein, albeit with different efficacies. The use of mice transgenic for the human HLA-A2.1 molecule indicated that the rSFV-expressed NS3 protein induces, as shown previously for an NS3 DNA vaccine, NS3-specific cytotoxic lymphocytes (CTLs) targeted at one dominant HLA-A2 epitope described in infected patients. All DNA/rSFV vaccine combinations evaluated induced specific CTLs, which were detectable for up to 31 weeks after the first injection. Overall, less than 1 log difference was observed in terms of the vigour of the bulk CTL response induced and the CTL precursor frequency between all vaccines (ranging from 1:2·6×105 to 1:1×106). Anti-NS3 antibodies could only be detected following a combined vaccine regimen in non-transgenic BALB/c mice. In conclusion, rSFV particles expressing NS3 are capable of inducing NS3-specific cellular immune responses targeted at a major HLA-A2 epitope. Such responses were comparable to those obtained with a DNA-based NS3 vaccine, whether in the context of single or combined regimens.

scholarly journals Genetic Analysis of Hepatitis C Virus with Defective Genome and Its Infectivity in Vitro

2009 ◽  
Vol 83 (13) ◽  
pp. 6922-6928 ◽  
Author(s):  
Kazuo Sugiyama ◽  
Kenji Suzuki ◽  
Takahide Nakazawa ◽  
Kenji Funami ◽  
Takayuki Hishiki ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Genetic Analysis ◽  
Phylogenetic Analyses ◽  
Structural Proteins ◽  
Infectious Particle ◽  
First Time ◽  
Self Replication

ABSTRACT Replication and infectivity of hepatitis C virus (HCV) with a defective genome is ambiguous. We molecularly cloned 38 HCV isolates with defective genomes from 18 patient sera. The structural regions were widely deleted, with the 5′ untranslated, core, and NS3-NS5B regions preserved. All of the deletions were in frame, indicating that they are translatable to the authentic terminus. Phylogenetic analyses showed self-replication of the defective genomes independent of full genomes. We generated a defective genome of chimeric HCV to mimic the defective isolate in the serum. By using this, we demonstrated for the first time that the defective genome, as it is circulating in the blood, can be encapsidated as an infectious particle by trans complementation of the structural proteins.

scholarly journals Differences in the expression of the hepatitis C virus core+1 open reading frame between a nuclear and a cytoplasmic expression system

2008 ◽  
Vol 89 (1) ◽  
pp. 222-231 ◽  
Author(s):  
Niki Vassilaki ◽  
Katerina I. Kalliampakou ◽  
Penelope Mavromara
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mammalian Cells ◽  
Expression System ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Transcription System ◽  
The Core ◽  
Internal Core

The hepatitis C virus (HCV) genome possesses an open reading frame (ORF) overlapping the core gene at +1 nucleotide (core+1 ORF). Initial in vitro studies suggested that the core+1 ORF is translated by a ribosomal −2/+1 frameshift mechanism during elongation of the viral polyprotein. Recent studies, however, based on transfection of mammalian cells with reporter constructs have shown that translation of the core+1 ORF is mediated from internal core+1 codons. To resolve the apparent discrepancies associated with the mechanism of core+1 translation, we examined the expression of the HCV-1 and HCV-1a (H) core+1 ORF in a cytoplasmic transcription system based on Huh-7/T7 cells that constitutively synthesize the T7 RNA polymerase in comparison to that in Huh-7 cells. We showed that the efficiency of both the −2/+1 and −1/+2 frameshift events operating at the HCV-1 core codons 8–11 is significantly enhanced in the Huh-7/T7 cytoplasmic transcription system and is dependent on the presence of the consecutive adenine (A) residues within core codons 8–11. In contrast, internal translation initiation at core+1 codons 85/87 occurs in both the nuclear and cytoplasmic transcription systems and is not repressed by the ribosomal frameshifting event. Finally, although core+1 codons 85/87 is the most efficient site for internal initiation of core+1 translation, it may not be unique, as additional internal core+1 codon(s) appear to drive translation at low levels.

scholarly journals Functional analysis of hepatitis C virus E2 glycoproteins and virus-like particles reveals structural dissimilarities between different forms of E2

2001 ◽  
Vol 82 (8) ◽  
pp. 1877-1883 ◽  
Author(s):  
Ania Owsianka ◽  
Reginald F. Clayton ◽  
Lawrence D. Loomis-Price ◽  
Jane A. McKeating ◽  
Arvind H. Patel
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Structure Function ◽  
Mammalian Cells ◽  
Function Analysis ◽  
Virus Like Particles ◽  
Binding Characteristics ◽  
Virus Interaction ◽  
Cd81 Binding

Structure–function analysis of the hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, has been difficult due to the unavailability of HCV virions. Truncated soluble forms of E2 have been used as models to study virus interaction with the putative HCV receptor CD81, but they may not fully mimic E2 structures on the virion. Here, we compared the CD81-binding characteristics of truncated E2 (E2660) and full-length (FL) E1E2 complex expressed in mammalian cells, and of HCV virus-like particles (VLPs) generated in insect cells. All three glycoprotein forms interacted with human CD81 in an in vitro binding assay, allowing us to test a panel of well-characterized anti-E2 monoclonal antibodies (MAbs) for their ability to inhibit the glycoprotein–CD81 interaction. MAbs specific for E2 amino acid (aa) regions 396–407, 412–423 and 528–535 blocked binding to CD81 of all antigens tested. However, MAbs specific for regions 432–443, 436–443 and 436–447 inhibited the interaction of VLPs, but not of E2660 or the FL E1E2 complex with CD81, indicating the existence of structural differences amongst the E2 forms. These findings underscore the need to carefully select an appropriate ligand for structure–function analysis.

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