title: Comprehensive linker-scanning mutagenesis of the hepatitis C virus E1 and E2 envelope glycoproteins reveals new structure-function relationships.
abstract: Despite extensive research, many details about the structure and functions of hepatitis C virus (HCV) glycoproteins E1 and E2 are not fully understood, and their crystal structure remains to be determined. We applied linker-scanning mutagenesis to generate a panel of 34 mutants, each containing an insertion of 5 aa at a random position within the E1E2 sequence. The mutated glycoproteins were analysed by using a range of assays to identify regions critical for maintaining protein conformation, E1E2 complex assembly, CD81 receptor binding, membrane fusion and infectivity. The results, while supporting previously published data, provide several interesting new findings. Firstly, insertion at amino acid 587 or 596 reduced E1E2 heterodimerization without affecting reactivity with some conformation-sensitive mAbs or with CD81, thus implicating these residues in glycoprotein assembly. Secondly, insertions within a conserved region of E2, between amino acid residues 611 and 631, severely disrupted protein conformation and abrogated binding of all conformation-sensitive antibodies, suggesting that the structural integrity of this region is critical for the correct folding of E2. Thirdly, an insertion at Leu-682 specifically affected membrane fusion, providing direct evidence that the membrane-proximal 'stem' of E2 is involved in the fusion mechanism. Overall, our results show that the HCV glycoproteins generally do not tolerate insertions and that there are a very limited number of sites that can be changed without dramatic loss of function. Nevertheless, we identified two E2 insertion mutants, at amino acid residues 408 and 577, that were infectious in the murine leukemia virus-based HCV pseudoparticle system.
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, 8 Church Street, Glasgow G11 5JR, UK.|Department of Molecular Virology, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Rychłowska
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, 8 Church Street, Glasgow G11 5JR, UK.
authors.forename: Ania M
authors.initials: AM
authors.lastname: Owsianka
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, CA 94304, USA.
authors.forename: Steven K H
authors.initials: SKH
authors.lastname: Foung
authors.affiliation: Inserm U1019, F-59019 Lille, France.|Université Lille Nord de France, F-59000 Lille, France.|Institut Pasteur de Lille, Centre for Infection & Immunity of Lille (CIIL), F-59019 Lille, France.|CNRS UMR8204, F-59021 Lille, France.
authors.forename: Jean
authors.initials: J
authors.lastname: Dubuisson
authors.affiliation: Department of Molecular Virology, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland.
authors.forename: Krystyna
authors.initials: K
authors.lastname: Bieńkowska-Szewczyk
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, 8 Church Street, Glasgow G11 5JR, UK.
authors.forename: Arvind H
authors.initials: AH
authors.lastname: Patel
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D008561:Membrane Fusion; D008958:Models, Molecular; D016254:Mutagenesis, Insertional / Q000235:genetics* / Q000379:methods; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D011991:Receptors, Virus / Q000378:metabolism; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D015703:Antigens, CD; C554798:CD81 protein, human; C087035:E1 protein, Hepatitis C virus; D011991:Receptors, Virus; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus