Use of pseudotyped retroviral vectors to analyze the receptor-binding pocket of hemagglutinin from a pathogenic avian influenza A virus (H7 subtype).
The hemagglutinin (HA) protein of influenza virus binds to terminal sialic acid residues present on cell surface glycoproteins and glycolipids. The specific amino acids involved in this interaction have been identified for a H3 subtype HA from the human non-pathogenic virus, A/Aichi/2/68, by both crystallographic and mutagenesis studies. We were interested to examine the receptor-binding pocket of a H7 subtype protein from the avian pathogenic virus A/FPV/Rostock/34. Accordingly, we made amino acid substitutions at six conserved residues (Y88, T126, H174, E181, L185, and G219), suggested by comparison with the receptor-binding pocket of the H3 protein, and analyzed the resulting proteins using pseudotyped retroviral vectors. The use of these vectors enabled us to quantitate both the ability of the mutant HA proteins to bind with receptor-expressing cells, and also to promote virus-cell fusion by measuring vector titer. Using this system, we identified a subset of mutants with impaired receptor-binding activity and a corresponding decrease in titer, but which retained the ability to induce syncytia in low pH cell-cell fusion assays. The most severely affected mutants contained more than one substitution, with the triple mutant Y88F/E181Q/G219K being the most defective. These observations highlight the importance of multiple contact points for the interaction between sialic acid and HA.
Lin
Amy H
AH
Gene Therapy Laboratories, Norris Cancer Center, Room 6338, University of Southern California Keck School of Medicine, 1441 Eastlake Avenue, Los Angeles, CA 90033, USA.
Cannon
Paula M
PM
D016475:3T3 Cells; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D015726:Giant Cells; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009980:Influenza A virus / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity / Q000502:physiology; D008561:Membrane Fusion; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D011991:Receptors, Virus / Q000737:chemistry / Q000378:metabolism*; D012190:Retroviridae; D017386:Sequence Homology, Amino Acid; D012794:Sialic Acids / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011991:Receptors, Virus; D012794:Sialic Acids; C109963:hemagglutinin, avian influenza A virus