Human Monoclonal Antibody to Hepatitis C Virus E1 Glycoprotein That Blocks Virus Attachment and Viral Infectivity

Keck, Zhen Yong; Sung, Vicky M.-H.; Perkins, Susan; Rowe, Judy; Paul, Sudhir; Liang, T. Jake; Lai, Michael M. C.; Foung, Steven K. H.

doi:10.1128/jvi.78.13.7257-7263.2004

Cited by 96 publications

(100 citation statements)

References 19 publications

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“…Interestingly, the protonated peptide ion of m/z 656.66 resulted from the complete loss of the oligosaccharide by glycosidic bond cleavage between the sugar and the Asn side chain. Unlike the other MS/MS data, a large number of specific backbone cleavages were observed (b 3 , b 4 , b 5 , and y 4 ) and the peptide backbone could be almost completely assigned based on this spectrum (Figure 4b). In addition, backbone fragments still containing one or two GlcNAc N N units were abundant.…”

Section: Resultsmentioning

confidence: 93%

“…The two envelope proteins E1 and E2 are heavily N-glycosylated and are believed to be Type 1 transmembrane protein with N terminal ectodomains and C-terminal hydrophobic anchors [3]. Together, they are expected to form the viral envelope [4]. During their synthesis, the ectodomains of HCV glycoproteins are targeted to the ER lumen, where they are modified by N-linked glycosylation.…”

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confidence: 99%

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Mass spectrometric characterization of glycosylation of hepatitis C virus E2 envelope glycoprotein reveals extended microheterogeneity of N-glycans

Iacob

Perdivara

Przybylski

et al. 2008

J. Am. Soc. Mass Spectrom.

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Hepatitis C virus (HCV) causes acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The polyprotein encoded in the HCV genome is co-and post-translationally processed by host and viral peptidases, generating the structural proteins Core, E1, E2, and p7, and five nonstructural proteins. The two envelope proteins E1 and E2 are heavily glycosylated. Studying the glycan moieties attached to the envelope E2 glycoprotein is important because the N-linked glycans on E2 envelope protein are involved in the interaction with some human neutralizing antibodies, and may also have a direct or indirect effect on protein folding. In the present study, we report the mass spectrometric characterization of the glycan moieties attached to the E2 glycoprotein. The mass spectrometric analysis clearly identified the nature, composition, and microheterogeneity of the sugars attached to the E2 glycopeptides. All 11 sites of glycosylation on E2 protein were characterized, and the majority of these sites proved to be occupied by high mannose glycans. However, complex type oligosaccharides, which have not been previously identified, were exclusively observed at two N-linked sites, and their identity and heterogeneity were determined. (J

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Section: Resultsmentioning

confidence: 93%

mentioning

confidence: 99%

Mass spectrometric characterization of glycosylation of hepatitis C virus E2 envelope glycoprotein reveals extended microheterogeneity of N-glycans

Iacob

Perdivara

Przybylski

et al. 2008

J. Am. Soc. Mass Spectrom.

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show abstract

“…These assays were carried out in solid phase, with HCVpp bound to lectincoated microtiter plates, and bound mAbs detected by an alkaline phosphatase-conjugated secondary antibody (see Materials and Methods). Results were normalized according to the HCVpp abundance in each preparation, which was determined by measuring the binding of a non-neutralizing human mAb (H-111) that recognizes a linear epitope within E1 (29). These studies revealed complete loss of AP33 binding to the N415Y and N415YϩE655G mutants (Fig.…”

Section: Resultsmentioning

confidence: 99%

In vitro selection of a neutralization-resistant hepatitis C virus escape mutant

Gal-Tanamy

Keck

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Effective immunization against hepatitis C virus (HCV) infections is likely to require the induction of both robust T and B cell immunity. Although neutralizing antibodies may play an important role in control of infection, there is little understanding of the structure of the HCV envelope glycoproteins and how they interact with such antibodies. An additional challenge for vaccine design is the genetic diversity of HCV and the rapid evolution of viral quasispecies that escape antibody-mediated neutralization. We used a cell culture-infectious, chimeric HCV with the structural proteins of genotype 1a virus to identify envelope residues contributing to the epitope recognized by a broadly neutralizing, murine monoclonal antibody, AP33. By repetitive rounds of neutralization followed by amplification, we selected a population of viral escape mutants that resist stringent neutralization with AP33 and no longer bind the antibody. Two amino acid substitutions, widely separated in the linear sequence of the E2 envelope protein (N415Y and E655G), were identified by sequencing of cloned cDNA and shown by reverse genetics analysis to contribute jointly to the AP33 resistance phenotype. The N415Y mutation substantially lowered virus fitness, most likely because of a defect in viral entry, but did not reduce binding of soluble CD81 to immobilized HCV-pseudotyped retrovirus particles. The in vitro selection of an HCV escape mutant recapitulates the ongoing evolution of antigenic variants that contributes to viral persistence in humans and reveals information concerning the conformational structure of the AP33 epitope, its role in viral replication, and constraints on its molecular evolution.antibody ͉ immunity ͉ vaccine ͉ viral envelope

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“…However, these systems are not easy to use to study viral fusion in vitro, because of safety restrictions and because of the fact that wild type HCV particle production is tightly coupled to viral replication, a process that is very sensitive to mutations or alterations of the genomic sequence. We and others have shown previously that HCVpp, a model system for HCV cell entry based on retroviral or lentiviral core particles displaying HCV E1 and E2, closely mimic the early entry steps of the wild type virus (12)(13)(14)(15)(16)(17)(18)(19). Indeed, HCVpp display a preferential tropism for hepatic cells, require the presence of both glycoproteins for activity, and can be neutralized by HCV patient sera.…”

Section: Hepatitis C Virus (Hcv)mentioning

confidence: 99%

Hepatitis C Virus Glycoproteins Mediate Low pH-dependent Membrane Fusion with Liposomes

Lavillette

Bartosch

Nourrisson

et al. 2006

Journal of Biological Chemistry

126

176

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It has been suggested that the hepatitis C virus (HCV) infects host cells through a pH-dependent internalization mechanism, but the steps leading from virus attachment to the fusion of viral and cellular membranes remain uncharacterized. Here we studied the mechanism underlying the HCV fusion process in vitro using liposomes and our recently described HCV pseudoparticles (pp) bearing functional E1E2 envelope glycoproteins. The fusion of HCVpp with liposomes was monitored with fluorescent probes incorporated into either the HCVpp or the liposomes. To validate these assays, pseudoparticles bearing either the hemagglutinin of the influenza virus or the amphotropic glycoprotein of murine leukemia virus were used as models for pH-dependent and pH-independent entry, respectively. The use of assays based either on fusion-induced dequenching of fluorescent probes or on reporter systems, which produce fluorescence when the virus and liposome contents are mixed, allowed us to demonstrate that HCVpp mediated a complete fusion process, leading to the merging of both membrane leaflets and to the mixing of the internal contents of pseudoparticle and liposome. This HCVpp-mediated fusion was dependent on low pH, with a threshold of 6.3 and an optimum at about 5.5. Fusion was temperature-dependent and did not require any protein or receptor at the surface of the target liposomes. Most interestingly, fusion was facilitated by the presence of cholesterol in the target membrane. These findings clearly indicate that HCV infection is mediated by a pH-dependent membrane fusion process. This paves the way for future studies of the mechanisms underlying HCV membrane fusion. Hepatitis C virus (HCV)2 belongs to the genus Hepacivirus of the Flaviviridae family (1) and has infected some 170 million people worldwide (2). In the majority of HCV-infected patients, the progression to chronic disease is associated with an increased risk of liver diseases and hepatocellular carcinoma. No vaccine is presently available, and the current treatment for chronic hepatitis C (a combination of pegylated interferon and ribavirin) has a limited efficacy (3). A more detailed understanding of the molecular mechanisms of virus entry would be beneficial to the development of novel therapeutic strategies.The HCV genome is a positive-stranded RNA encoding a precursor polyprotein of about 3,000 amino acids. This polyprotein is cleaved coand post-translationally to generate 10 viral proteins, classified into structural (capsid protein and the envelope glycoproteins E1 and E2) and nonstructural proteins (NS2 to NS5B), separated by the membrane ion channel polypeptide p7 (1, 4). The type I transmembrane envelope glycoproteins E1 and E2 form noncovalently linked heterodimers in the endoplasmic reticulum and are highly glycosylated, containing up to 6 and 11 potential glycosylation sites, respectively (5-8). Studies of the HCV life cycle have been hampered by the lack of an efficient and reliable cell culture system to produce and isolate the functional virus. Most re...

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Human Monoclonal Antibody to Hepatitis C Virus E1 Glycoprotein That Blocks Virus Attachment and Viral Infectivity

Cited by 96 publications

References 19 publications

Mass spectrometric characterization of glycosylation of hepatitis C virus E2 envelope glycoprotein reveals extended microheterogeneity of N-glycans

Mass spectrometric characterization of glycosylation of hepatitis C virus E2 envelope glycoprotein reveals extended microheterogeneity of N-glycans

In vitro selection of a neutralization-resistant hepatitis C virus escape mutant

Hepatitis C Virus Glycoproteins Mediate Low pH-dependent Membrane Fusion with Liposomes

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