scholarly journals Expressionof Unmodified Hepatitis C Virus Envelope Glycoprotein-Coding SequencesLeads to Cryptic Intron Excision and Cell Surface Expression of E1/E2Heterodimers Comprising Full-Length and Partially DeletedE1

2003 ◽  
Vol 77 (24) ◽  
pp. 13418-13424 ◽  
Author(s):  
Julie Dumonceaux ◽  
Emmanuel G. Cormier ◽  
Francis Kajumo ◽  
Gerald P. Donovan ◽  
Jayanta Roy-Chowdhury ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Virus ◽  
Expression System ◽  
Surface Expression ◽  
Viral Envelope ◽  
Cell Surface Expression ◽  
Envelope Glycoproteins ◽  
Virus Envelope ◽  
Positive Strand Rna

ABSTRACT Hepatitis C virus (HCV) is a positive-strand RNA virus that replicates exclusively in the cytoplasm of infected cells. The viral envelope glycoproteins, E1 and E2, appear to be retained in the endoplasmic reticulum, where viral budding is thought to occur. Surprisingly, we found that the expression system used to generate HCV envelope glycoproteins influences their subcellular localization and processing. These findings have important implications for optimizing novel HCV fusion and entry assays as well as for budding and virus particle formation.

scholarly journals Role of N-Linked Glycans in the Functions of Hepatitis C Virus Envelope Glycoproteins

2005 ◽  
Vol 79 (13) ◽  
pp. 8400-8409 ◽  
Author(s):  
Anne Goffard ◽  
Nathalie Callens ◽  
Birke Bartosch ◽  
Czeslaw Wychowski ◽  
François-Loïc Cosset ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Glycosylation Site ◽  
Viral Envelope ◽  
Envelope Glycoproteins ◽  
Wild Type ◽  
Virus Envelope ◽  
Glycosylation Sites

ABSTRACT Hepatitis C virus (HCV) encodes two viral envelope glycoproteins. E1 contains 4 or 5 N-linked glycosylation sites and E2 contains up to 11, with most of the sites being well conserved, suggesting that they play an essential role in some functions of these proteins. For this study, we used retroviral pseudotyped particles harboring mutated HCV envelope glycoproteins to study these glycans. The mutants were named with an N followed by a number related to the relative position of the potential glycosylation site in each glycoprotein (E1N1 to E1N4 for E1 mutants and E2N1 to E2N11 for E2 mutants). The characterization of these mutants allowed us to define three phenotypes. For the first group (E1N3, E2N3, E2N5, E2N6, E2N7, and E2N9), the infectivities of the mutants were close to that of the wild type. The second group (E1N1, E1N2, E1N4, E2N1, and E2N11) contained mutants that were still infectious but whose infectivities were reduced to <50% that of the wild type. The third group (E2N2, E2N4, E2N8, and E2N10) contained mutants that had almost totally lost infectivity. The absence of infectivity of the E2N8 and E2N10 mutants was due to the lack of incorporation of the E1E2 heterodimer into HCVpp, which was due to misfolding of the heterodimer, as shown by immunoprecipitation with conformation-sensitive antibodies and by a CD81 pull-down assay. The absence of infectivity of the E2N2 and E2N4 mutants indicated that these two glycans are involved in controlling HCV entry. Altogether, the data indicate that some glycans of HCV envelope glycoproteins play a major role in protein folding and others play a role in HCV entry.

scholarly journals Characterization of Functional Hepatitis C Virus Envelope Glycoproteins

2004 ◽  
Vol 78 (6) ◽  
pp. 2994-3002 ◽  
Author(s):  
Anne Op De Beeck ◽  
Cécile Voisset ◽  
Birke Bartosch ◽  
Yann Ciczora ◽  
Laurence Cocquerel ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Conformational Changes ◽  
Structural Changes ◽  
Envelope Proteins ◽  
Viral Envelope ◽  
Envelope Glycoproteins ◽  
Functional Envelope

ABSTRACT Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2, that assemble as a noncovalent heterodimer which is mainly retained in the endoplasmic reticulum. Because assembly into particles and secretion from the cell lead to structural changes in viral envelope proteins, characterization of the proteins associated with the virion is necessary in order to better understand how they mature to be functional in virus entry. There is currently no efficient and reliable cell culture system to amplify HCV, and the envelope glycoproteins associated with the virion have therefore not been characterized yet. Recently, infectious pseudotype particles that are assembled by displaying unmodified HCV envelope glycoproteins on retroviral core particles have been successfully generated. Because HCV pseudotype particles contain fully functional envelope glycoproteins, these envelope proteins, or at least a fraction of them, should be in a mature conformation similar to that on the native HCV particles. In this study, we used conformation-dependent monoclonal antibodies to characterize the envelope glycoproteins associated with HCV pseudotype particles. We showed that the functional unit is a noncovalent E1E2 heterodimer containing complex or hybrid type glycans. We did not observe any evidence of maturation by a cellular endoprotease during the transport of these envelope glycoproteins through the secretory pathway. These envelope glycoproteins were recognized by a panel of conformation-dependent monoclonal antibodies as well as by CD81, a molecule involved in HCV entry. The functional envelope glycoproteins associated with HCV pseudotype particles were also shown to be sensitive to low-pH treatment. Such conformational changes are likely necessary to initiate fusion.

330 MOLECULAR SIGNATURES OF HEPATITIS C VIRUS ENVELOPE GLYCOPROTEINS AND RESPONSE TO ANTIVIRAL TREATMENT

2009 ◽  
Vol 50 ◽  
pp. S128
Author(s):  
R. Moenne-Loccoz ◽  
C. Rajafinjatovo ◽  
S. Fafi-Kremer ◽  
F. Habersetzer ◽  
A. Ananna ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Treatment ◽  
Envelope Glycoproteins ◽  
Molecular Signatures ◽  
Virus Envelope

Mapping and characterization of B cell linear epitopes in the conservative regions of hepatitis C virus envelope glycoproteins

2002 ◽  
Vol 9 (3) ◽  
pp. 174-182 ◽  
Author(s):  
L. V. Olenina ◽  
L. I. Nikolaeva ◽  
B. N. Sobolev ◽  
N. P. Blokhina ◽  
A. I. Archakov ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
B Cell ◽  
Envelope Glycoproteins ◽  
Virus Envelope ◽  
Linear Epitopes

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

2009 ◽  
Vol 83 (21) ◽  
pp. 11078-11089 ◽  
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.

B-Cell Linear Epitopes in the Conservative Regions of Hepatitis C Virus Envelope Glycoproteins

2001 ◽  
pp. 1031-1032
Author(s):  
Ekaterina F. Kolesanova ◽  
Ludmila V. Olenina ◽  
Boris N. Sobolev ◽  
Ludmila I. Nikolaeva ◽  
Alexander I. Archakov
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
B Cell ◽  
Envelope Glycoproteins ◽  
Virus Envelope ◽  
Linear Epitopes

scholarly journals Characterization of Antibodies Induced by Vaccination with Hepatitis C Virus Envelope Glycoproteins

2010 ◽  
Vol 202 (6) ◽  
pp. 862-866 ◽  
Author(s):  
Ranjit Ray ◽  
Keith Meyer ◽  
Arup Banerjee ◽  
Arnab Basu ◽  
Stephen Coates ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Envelope Glycoproteins ◽  
Virus Envelope

scholarly journals The Level of CD81 Cell Surface Expression Is a Key Determinant for Productive Entry of Hepatitis C Virus into Host Cells

2006 ◽  
Vol 81 (2) ◽  
pp. 588-598 ◽  
Author(s):  
George Koutsoudakis ◽  
Eva Herrmann ◽  
Stephanie Kallis ◽  
Ralf Bartenschlager ◽  
Thomas Pietschmann
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Surface ◽  
Surface Expression ◽  
Hcv Infection ◽  
Host Cells ◽  
Productive Infection ◽  
Cell Surface Expression ◽  
Type I ◽  
Cell Clones

ABSTRACT Recently a cell culture model supporting the complete life cycle of the hepatitis C virus (HCV) was developed. Searching for host cell determinants involved in the HCV replication cycle, we evaluated the efficiency of virus propagation in different Huh-7-derived cell clones. We found that Huh-7.5 cells and Huh7-Lunet cells, two former replicon cell clones that had been generated by removal of an HCV replicon by inhibitor treatment, supported comparable levels of RNA replication and particle production, whereas virus spread was severely impaired in the latter cells. Analysis of cell surface expression of CD81 and scavenger receptor class B type I (SR-BI), two molecules previously implicated in HCV entry, revealed similar expression levels for SR-BI, while CD81 surface expression was much higher on Huh-7.5 cells than on Huh7-Lunet cells. Ectopic expression of CD81 in Huh7-Lunet cells conferred permissiveness for HCV infection to a level comparable to that for Huh-7.5 cells. Modulation of CD81 cell surface density in Huh-7.5 cells by RNA interference indicated that a certain amount of this molecule (∼7 × 104 molecules per cell) is required for productive infection with a low dose of HCV. Consistent with this, we show that susceptibility to HCV infection depends on a critical quantity of CD81 molecules. While infection is restricted in cells expressing very small amounts of CD81, susceptibility rapidly rises within a narrow range of CD81 levels, reaching a plateau where higher expression does not further increase the efficiency of infection. Together these data indicate that a high density of cell surface-exposed CD81 is a key determinant for productive HCV entry into host cells.

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