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.

scholarly journals Analysis of Antigenicity and Topology of E2 Glycoprotein Present on Recombinant Hepatitis C Virus-Like Particles

2002 ◽  
Vol 76 (15) ◽  
pp. 7672-7682 ◽  
Author(s):  
Reginald F. Clayton ◽  
Ania Owsianka ◽  
Jim Aitken ◽  
Susan Graham ◽  
David Bhella ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mammalian Cells ◽  
Function Analysis ◽  
Recombinant Baculovirus ◽  
Microscopic Analysis ◽  
Surface Topology ◽  
Viral Glycoprotein ◽  
Electron Microscopic ◽  
Virus Like Particles

ABSTRACT Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2660) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2660 with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2660 but not VLPs or FL E1E2 complex. The antigenic differences between E2660, FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.

scholarly journals Structure-Function Analysis of Hepatitis C Virus Envelope-CD81 Binding

2000 ◽  
Vol 74 (10) ◽  
pp. 4824-4830 ◽  
Author(s):  
R. Petracca ◽  
F. Falugi ◽  
G. Galli ◽  
N. Norais ◽  
D. Rosa ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Structure Function ◽  
Function Analysis ◽  
Virus Envelope ◽  
Cd81 Binding

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 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.

A C-terminal truncated hepatitis C virus core protein variant assembles in vitro into virus-like particles in the absence of structured nucleic acids

2005 ◽  
Vol 334 (3) ◽  
pp. 901-906 ◽  
Author(s):  
Nelson Acosta-Rivero ◽  
Armando Rodriguez ◽  
Alexis Mussachio ◽  
Johana Poutu ◽  
Viviana Falcon ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nucleic Acids ◽  
Core Protein ◽  
Protein Variant ◽  
Virus Like Particles ◽  
Virus Core

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 Structural Features of Envelope Proteins on Hepatitis C Virus-like Particles as Determined by Anti-envelope Monoclonal Antibodies and CD81 Binding

Virology ◽  
2002 ◽  
Vol 298 (1) ◽  
pp. 124-132 ◽  
Author(s):  
Miriam Triyatni ◽  
John Vergalla ◽  
Anthony R. Davis ◽  
Kenneth G. Hadlock ◽  
Steven K.H. Foung ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Structural Features ◽  
Envelope Proteins ◽  
Virus Like Particles ◽  
Cd81 Binding
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