title: Construction and characterization of chimeric hepatitis C virus E2 glycoproteins: analysis of regions critical for glycoprotein aggregation and CD81 binding.
abstract: We compared the ability of two closely related truncated E2 glycoproteins (E2(660)) derived from hepatitis C virus (HCV) genotype 1a strains Glasgow (Gla) and H77c to bind a panel of conformation-dependent monoclonal antibodies (MAbs) and CD81. In contrast to H77c, Gla E2(660) formed disulfide-linked high molecular mass aggregates and failed to react with conformation-dependent MAbs and CD81. To delineate amino acid (aa) regions associated with protein aggregation and CD81 binding, several Gla-H77c E2(660) chimeric glycoproteins were constructed. Chimeras C1, C2 and C6, carrying aa 525-660 of Gla E2(660), produced disulfide-linked aggregates and failed to bind CD81 and conformation-dependent MAbs, suggesting that amino acids within this region are responsible for protein misfolding. The presence of Gla hypervariable region 1 (aa 384-406) on H77 E2(660), chimera C4, had no effect on protein folding or CD81 binding. Chimeras C3 and C5, carrying aa 384-524 or 407-524 of Gla E2(660), respectively, were recognized by conformation-dependent MAbs and yet failed to bind CD81, suggesting that amino acids in region 407-524 are important in modulating CD81 interaction without affecting antigen folding. Comparison of Gla and H77c E2(660) aa sequences with those of genotype 1a and divergent genotypes identified a number of variant amino acids, including two putative N-linked glycosylation sites at positions 476 and 532. However, introduction of G476N-G478S and/or D532N in Gla E2(660) had no effect on antigenicity or aggregation.
authors.affiliation: MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK1.
authors.forename: Arvind H
authors.initials: AH
authors.lastname: Patel
authors.affiliation: MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK1.
authors.forename: Jonny
authors.initials: J
authors.lastname: Wood
authors.affiliation: Institut de Biologie et Chimie des Protéines, UPR 412 CNRS, 7 Passage du Vercors, F-69367 Lyon Cedex 07, France2.
authors.forename: Francois
authors.initials: F
authors.lastname: Penin
authors.affiliation: CNRS-UMR8526, IBL/Institut Pasteur de Lille, 59021 Lille Cedex, France3.
authors.forename: Jean
authors.initials: J
authors.lastname: Dubuisson
authors.affiliation: University of Reading, School of Animal & Microbial Sciences, PO Box 228, Reading, UK4.
authors.forename: J A
authors.initials: JA
authors.lastname: McKeating
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D015703:Antigens, CD / Q000378:metabolism*; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D004220:Disulfides / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D005434:Flow Cytometry; D005838:Genotype; D006031:Glycosylation; D016174:Hepacivirus / Q000737:chemistry / Q000145:classification / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D011110:Polymorphism, Genetic / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D012995:Solubility; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D015703:Antigens, CD; D000956:Antigens, Viral; C554798:CD81 protein, human; D004220:Disulfides; D000939:Epitopes; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins