title: A conserved Gly436-Trp-Leu-Ala-Gly-Leu-Phe-Tyr motif in hepatitis C virus glycoprotein E2 is a determinant of CD81 binding and viral entry.
abstract: The hepatitis C virus (HCV) glycoproteins E1 and E2 form a heterodimer that mediates CD81 receptor binding and viral entry. In this study, we used site-directed mutagenesis to examine the functional role of a conserved G436WLAGLFY motif of E2. The mutants could be placed into two groups based on the ability of mature virion-incorporated E1E2 to bind the large extracellular loop (LEL) of CD81 versus the ability to mediate cellular entry of pseudotyped retroviral particles. Group 1 comprised E2 mutants where LEL binding ability largely correlated with viral entry ability, with conservative and nonconservative substitutions (W437 L/A, L438A, L441V/F, and F442A) inhibiting both functions. These data suggest that Trp-437, Leu-438, Leu-441, and Phe-442 directly interact with the LEL. Group 2 comprised E2 glycoproteins with more conservative substitutions that lacked LEL binding but retained between 20% and 60% of wild-type viral entry competence. The viral entry competence displayed by group 2 mutants was explained by residual binding by the E2 receptor binding domain to cellular full-length CD81. A subset of mutants maintained LEL binding ability in the context of intracellular E1E2 forms, but this function was largely lost in virion-incorporated glycoproteins. These data suggest that the CD81 binding site undergoes a conformational transition during glycoprotein maturation through the secretory pathway. The G436P mutant was an outlier, retaining near-wild-type levels of CD81 binding but lacking significant viral entry ability. These findings indicate that the G436WLAGLFY motif of E2 functions in CD81 binding and in pre- or post-CD81-dependent stages of viral entry.
authors.affiliation: The Macfarlane Burnet Institute for Medical Research and Public Health Ltd., GPO Box 2284, Melbourne, Victoria, Australia 3001. hdrummer@burnet.edu.au
authors.forename: Heidi E
authors.initials: HE
authors.lastname: Drummer
authors.forename: Irene
authors.initials: I
authors.lastname: Boo
authors.forename: Anne L
authors.initials: AL
authors.lastname: Maerz
authors.forename: Pantelis
authors.initials: P
authors.lastname: Poumbourios
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D015703:Antigens, CD / Q000378:metabolism*; D017124:Conserved Sequence; D019281:Dimerization; D016174:Hepacivirus / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011487:Protein Conformation; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D015703:Antigens, CD; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus