title: Distinct roles in folding, CD81 receptor binding and viral entry for conserved histidine residues of hepatitis C virus glycoprotein E1 and E2.
abstract: The protonation of histidine in acidic environments underpins its role in regulating the function of pH-sensitive proteins. For pH-sensitive viral fusion proteins, histidine protonation in the endosome leads to the activation of their membrane fusion function. The HCV (hepatitis C virus) glycoprotein E1-E2 heterodimer mediates membrane fusion within the endosome, but the roles of conserved histidine residues in the formation of a functional heterodimer and in sensing pH changes is unknown. We examined the functional roles of conserved histidine residues located within E1 and E2. The E1 mutations, H222A/R, H298R and H352A, disrupted E1-E2 heterodimerization and reduced virus entry. A total of five out of six histidine residues located within the E2 RBD (receptor-binding domain) were important for the E2 fold, and their substitution with arginine or alanine caused aberrant heterodimerization and/or CD81 binding. Distinct roles in E1-E2 heterodimerization and in virus entry were identified for His691 and His693 respectively within the membrane-proximal stem region. Viral entry and cell-cell fusion at neutral and low pH values were enhanced with H445R, indicating that the protonation state of His445 is a key regulator of HCV fusion. However, H445R did not overcome the block to virus entry induced by bafilomycin A1, indicating a requirement for an endosomal activation trigger in addition to acidic pH.
authors.affiliation: Macfarlane Burnet Institute for Medical Research and Public Health, 85 Commerical Road, Melbourne, Victoria 3004, Australia.
authors.forename: Irene
authors.initials: I
authors.lastname: Boo
authors.forename: Kevin
authors.initials: K
authors.lastname: teWierik
authors.forename: Florian
authors.initials: F
authors.lastname: Douam
authors.forename: Dimitri
authors.initials: D
authors.lastname: Lavillette
authors.forename: Pantelis
authors.initials: P
authors.lastname: Poumbourios
authors.forename: Heidi E
authors.initials: HE
authors.lastname: Drummer
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017124:Conserved Sequence; D057809:HEK293 Cells; D016174:Hepacivirus / Q000472:pathogenicity / Q000502:physiology*; D006639:Histidine / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D060225:Tetraspanin 28 / Q000737:chemistry*; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014771:Virion; D053586:Virus Internalization
chemical_list: C554798:CD81 protein, human; C087035:E1 protein, Hepatitis C virus; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus; D006639:Histidine