Analysis of Antigenicity and Topology of E2 Glycoprotein Present on Recombinant Hepatitis C 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.

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

Journal of General Virology ◽

10.1099/0022-1317-82-8-1877 ◽

2001 ◽

Vol 82 (8) ◽

pp. 1877-1883 ◽

Cited By ~ 140

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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Hepatitis C virus structural proteins and virus-like particles produced in recombinant baculovirus-infected insect cells

Molecular Biology ◽

10.1134/s0026893310010139 ◽

2010 ◽

Vol 44 (1) ◽

pp. 97-108 ◽

Cited By ~ 5

Author(s):

S. N. Belzhelarskaya ◽

N. N. Koroleva ◽

V. V. Popenko ◽

V. L. Drutza ◽

O. V. Orlova ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Insect Cells ◽

Recombinant Baculovirus ◽

Structural Proteins ◽

Virus Like Particles ◽

Infected Insect

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Cellular Binding and Entry of Hepatitis C Virus Pseudotypes and Virus-like Particles Requires a Different Set of Cellular Co-receptors

Zeitschrift für Gastroenterologie ◽

10.1055/s-2006-931747 ◽

2006 ◽

Vol 44 (01) ◽

Author(s):

A Haberstroh ◽

H Barth ◽

EK Schnober ◽

JM Pestka ◽

HM Diepolder ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Like Particles

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Control of PKR Protein Kinase by Hepatitis C Virus Nonstructural 5A Protein: Molecular Mechanisms of Kinase Regulation

Molecular and Cellular Biology ◽

10.1128/mcb.18.9.5208 ◽

1998 ◽

Vol 18 (9) ◽

pp. 5208-5218 ◽

Cited By ~ 431

Author(s):

Michael Gale ◽

Collin M. Blakely ◽

Bart Kwieciszewski ◽

Seng-Lai Tan ◽

Michelle Dossett ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protein Kinase ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Functional Assay ◽

Binding Region ◽

Dimerization Domain ◽

Antiviral Effects

ABSTRACT The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2α phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

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Efficient trans-Encapsidation of Hepatitis C Virus RNAs into Infectious Virus-Like Particles

Journal of Virology ◽

10.1128/jvi.00118-08 ◽

2008 ◽

Vol 82 (14) ◽

pp. 7034-7046 ◽

Cited By ~ 112

Author(s):

Eike Steinmann ◽

Christiane Brohm ◽

Stephanie Kallis ◽

Ralf Bartenschlager ◽

Thomas Pietschmann

Keyword(s):

Tissue Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

High Efficiency ◽

Infectious Virus ◽

Buoyant Density ◽

Target Cells ◽

Packaging Cell Line ◽

Virus Like Particles ◽

Rna Elements

ABSTRACT Recently, complete replication of hepatitis C virus (HCV) in tissue culture was established using the JFH1 isolate. To analyze determinants of HCV genome packaging and virion assembly, we developed a system that supports particle production based on trans-packaging of subgenomic viral RNAs. Using JFH1 helper viruses, we show that subgenomic JFH1 replicons lacking the entire core to NS2 coding region are efficiently encapsidated into infectious virus-like particles. Similarly, chimeric helper viruses with heterologous structural proteins trans-package subgenomic JFH1 replicons. Like authentic cell culture-produced HCV (HCVcc) particles, these trans-complemented HCV particles (HCVTCP) penetrate target cells in a CD81 receptor-dependent fashion. Since HCVTCP production was limited by competition between the helper and subgenomic RNA and to avoid contamination of HCVTCP stocks with helper viruses, we created HCV packaging cells. These cells encapsidate various HCV replicons with high efficiency, reaching infectivity titers up to 106 tissue culture infectious doses 50 per milliliter. The produced particles display a buoyant density comparable to HCVcc particles and can be propagated in the packaging cell line but support only a single-round infection in naïve cells. Together, this work demonstrates that subgenomic HCV replicons are assembly competent, thus excluding cis-acting RNA elements in the core-to-NS2 genomic region essential for RNA packaging. The experimental system described here should be helpful to decipher the mechanisms of HCV assembly and to identify RNA elements and viral proteins involved in particle formation. Similar to other vector systems of plus-strand RNA viruses, HCVTCP may prove valuable for gene delivery or vaccination approaches.

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Blocking Hepatitis C Virus Infection with Recombinant Form of Envelope Protein 2 Ectodomain

Journal of Virology ◽

10.1128/jvi.00800-09 ◽

2009 ◽

Vol 83 (21) ◽

pp. 11078-11089 ◽

Cited By ~ 43

Author(s):

Jillian Whidby ◽

Guaniri Mateu ◽

Hannah Scarborough ◽

Borries Demeler ◽

Arash Grakoui ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Mammalian Cells ◽

Expression System ◽

Developed Countries ◽

Viral Envelope ◽

Hcv Infection ◽

Size Exclusion ◽

Amino Terminal ◽

Endosomal Acidification

ABSTRACT More than 120 million people worldwide are chronically infected with hepatitis C virus (HCV), making HCV infection the leading cause of liver transplantation in developed countries. Treatment is limited, and efficacy depends upon the infecting strain and the initial viral load. The HCV envelope glycoproteins (E1 and E2) are involved in receptor binding, virus-cell fusion, and entry into the host cell. HCV infection proceeds by endosomal acidification, suggesting that fusion of the viral envelope with cellular membranes is a pH-triggered event. E2 consists of an amino-terminal ectodomain, an amphipathic helix that forms a stem region, and a carboxy-terminal membrane-associating segment. We have devised a novel expression system for the production of a secreted form of E2 ectodomain (eE2) from mammalian cells and performed a comprehensive biochemical and biophysical characterization. eE2 is properly folded, as determined by binding to human CD81, blocking of infection of cell culture-derived HCV, and recognition by antibodies from patients chronically infected with different genotypes of HCV. The glycosylation pattern, number of disulfide bonds, oligomerization state, and secondary structure of eE2 have been characterized using mass spectrometry, size exclusion chromatography, circular dichroism, and analytical ultracentrifugation. These results advance the understanding of E2 and may assist in the design of an HCV vaccine and entry inhibitor.

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