Ujike Makoto, Nakajima Katsuhisa, Nobusawa Eri
Department of Microbiology and Infection, Nagoya City University Graduate School of Medical Science, Kawasumi 1, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.
J Gen Virol. 2006 Jun;87(Pt 6):1669-1676. doi: 10.1099/vir.0.81528-0.
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.
乙型流感血凝素（BHA）细胞质尾（CT）的C末端序列由严格保守的疏水氨基酸组成，CT的最末端C末端氨基酸是亮氨酸（Leu）。为了阐明该氨基酸在BHA（B/神奈川/73）融合活性中的作用，通过将该位置的Leu替换为精氨酸（Arg）、赖氨酸（Lys）、丝氨酸（Ser）、色氨酸（Try）、缬氨酸（Val）或异亮氨酸（Ile），或完全删除Leu，创建了位点特异性突变体HA。所有突变体均在细胞表面表达，与红细胞结合，可正确切割成两个亚基，并且可以像野生型（wt）HA一样被酰化。通过脂质（R18）和水性（钙黄绿素）染料转移试验检测这些突变体的膜融合能力，并用合胞体形成试验进行定量。所有突变体HA对脂质混合或融合孔形成均未显示出可测量的影响。然而，C末端氨基酸疏水值低于Leu的突变体HA形成合胞体的能力降低，而具有更疏水氨基酸（Val或Ile）的突变体促进融合的程度与wt HA相当。另一方面，缺失Leu的突变体HA支持完全融合。这些结果表明，BHA-CT C末端最末端的Leu对于BHA介导的融合不是必需的，但该位置单个氨基酸的疏水性在合胞体形成中起重要作用。
