Studies on influenza haemagglutinin fusion peptide mutants generated by reverse genetics.
Influenza haemagglutinin (HA) is responsible for fusing viral and endosomal membranes during virus entry. In this process, conformational changes in the HA relocate the HA(2) N-terminal 'fusion peptide' to interact with the target membrane. The highly conserved HA fusion peptide shares composition and sequence features with functionally analogous regions of other viral fusion proteins, including the presence and distribution of glycines and large side-chain hydrophobic residues. HAs with mutations in the fusion peptide were expressed using vaccinia virus recombinants to examine the requirement for fusion of specific hydrophobic residues and the significance of glycine spacing. Mutant HAs were also incorporated into infectious influenza viruses for analysis of their effects on infectivity and replication. In most cases alanine, but not glycine substitutions for the large hydrophobic residues, yielded fusion-competent HAs and infectious viruses, suggesting that the conserved spacing of glycines may be structurally significant. When viruses containing alanine substitutions for large hydrophobic residues were passaged, pseudoreversion to valine was observed, indicating a preference for large hydrophobic residues at specific positions. Viruses were also obtained with serine, leucine or phenylalanine as the N-terminal residue, but these replicated to significantly lower levels than wild-type virus with glycine at this position.
Cross
K J
KJ
National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Wharton
S A
SA
Skehel
J J
JJ
Wiley
D C
DC
Steinhauer
D A
DA
D000143:Acids; D000818:Animals; D002460:Cell Line; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004285:Dogs; D015870:Gene Expression; D006367:HeLa Cells; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000502:physiology; D008561:Membrane Fusion / Q000502:physiology*; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000143:Acids; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D010455:Peptides; D011498:Protein Precursors