title: Amino acid residues in the fusion peptide pocket regulate the pH of activation of the H5N1 influenza virus hemagglutinin protein.
abstract: The receptor specificity and cleavability of the hemagglutinin (HA) protein have been shown to regulate influenza A virus transmissibility and pathogenicity, but little is known about how its pH of activation contributes to these important biological properties. To identify amino acid residues that regulate the acid stability of the HA protein of H5N1 influenza viruses, we performed a mutational analysis of the HA protein of the moderately pathogenic A/chicken/Vietnam/C58/04 (H5N1) virus. Nineteen HA proteins containing point mutations in the HA2 coiled-coil domain or in an HA1 histidine or basic patch were generated. Wild-type and mutant HA plasmids were transiently transfected in cell culture and analyzed for total protein expression, surface expression, cleavage efficiency, pH of fusion, and pH of conformational change. Four mutations to residues in the fusion peptide pocket, Y23H and H24Q in the HA1 subunit and E105K and N114K in the HA2 subunit, and a K58I mutation in the HA2 coiled-coil domain significantly altered the pH of activation of the H5 HA protein. In some cases, the magnitude and direction of changes of individual mutations in the H5 HA protein differed considerably from similar mutations in other influenza A virus HA subtypes. Introduction of Y23H, H24Q, K58I, and N114K mutations into recombinant viruses resulted in virus-expressed HA proteins with similar shifts in the pH of fusion. Overall, the data show that residues comprising the fusion peptide pocket are important in triggering pH-dependent activation of the H5 HA protein.
authors.affiliation: Department of Infectious Diseases, MS 330, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105-3678, USA.
authors.forename: Mark L
authors.initials: ML
authors.lastname: Reed
authors.forename: Hui-Ling
authors.initials: HL
authors.lastname: Yen
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: DuBois
authors.forename: Olga A
authors.initials: OA
authors.lastname: Bridges
authors.forename: Rachelle
authors.initials: R
authors.lastname: Salomon
authors.forename: Robert G
authors.initials: RG
authors.lastname: Webster
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Russell
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004252:DNA Mutational Analysis; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D053124:Influenza A Virus, H5N1 Subtype / Q000502:physiology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D014162:Transfection; D053586:Virus Internalization
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; C109963:hemagglutinin, avian influenza A virus