A point mutation at the C terminus of the cytoplasmic domain of influenza B virus haemagglutinin inhibits syncytium formation.
The C-terminal sequence of the cytoplasmic tail (CT) of influenza B haemagglutinin (BHA) consists of strictly conserved, hydrophobic amino acids, and the endmost C-terminal amino acid of the CT is Leu. To elucidate the role of this amino acid in the fusion activity of BHA (B/Kanagawa/73), site-specific mutant HAs were created by replacing Leu at this position with Arg, Lys, Ser, Try, Val or Ile or by the deletion of Leu altogether. All mutants were expressed at the cell surface, bound to red blood cells, were cleaved properly into two subunits and could be acylated like the wild-type (wt) HA. The membrane-fusion ability of these mutants was examined with a lipid (R18) and aqueous (calcein) dye-transfer assay and quantified with a syncytium-formation assay. All mutant HAs showed no measurable effect on lipid mixing or fusion-pore formation. However, mutant HAs with a hydrophobic value of the C-terminal amino acid lower than that of Leu had a reduced ability to form syncytia, whereas mutants with a more hydrophobic amino acid (Val or Ile) promoted fusion to the extent of the wt HA. On the other hand, the mutant HA with the deletion of Leu supported full fusion. These results demonstrate that Leu at the endmost portion of the C terminus of the BHA-CT is not essential for BHA-mediated fusion, but that the hydrophobicity of the single amino acid at this position plays an important role in syncytium formation.
Ujike
Makoto
M
Department of Microbiology and Infection, Nagoya City University Graduate School of Medical Science, Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.
Nakajima
Katsuhisa
K
Department of Microbiology and Infection, Nagoya City University Graduate School of Medical Science, Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.
Nobusawa
Eri
E
Department of Microbiology and Infection, Nagoya City University Graduate School of Medical Science, Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005434:Flow Cytometry; D015726:Giant Cells / Q000502:physiology*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D009981:Influenza B virus / Q000472:pathogenicity* / Q000502:physiology; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation
D019267:Hemagglutinin Glycoproteins, Influenza Virus