Nobusawa E, Hishida R, Murata M, Kawasaki K, Ohnishi S, Nakajima K
Department of Virology, Nagoya City University, Medical School, Japan.
Arch Virol. 1995;140(5):865-75. doi: 10.1007/BF01314963.
To clarify the role of acidic amino acid residues in the "fusion segment" of hemagglutinin (HA) of influenza A virus (H1N1) in pH-dependent membrane fusion, we have constructed and expressed five mutant HA cDNAs in CV-1 cells by SV40-HA virus vectors (SVHA). Fusion activities of the five mutant HAs were examined by lipid mixing and polykaryon formation assays. In spite of the substitution of Gly and Lys for the acidic residues, all the mutants were found to retain their low-pH-dependent fusion activity by lipid mixing assay. Although SVHA-G19(HA(2)19D-->G), -K11 (HA(2)11E-->K) and -K19(HA(2)19D-->K) induced polykaryon formation at low pH as wild type HA did, SVHA-G11(HA(2)11E-->G) induced limited polykaryon formation and SVHA-G11,19 (HA(2)11E-->G, 19D-->G) did not. The substitution of Gly for Glu at position 11 inhibited widening of the initial fusion pore. However, Lys mutants induced the formation of an initial fusion pore and widened it at low pH where Lys residues might have positive charges. These results suggest that the neutralization of the charges on acidic residues in the "fusion segment" at low pH is not important for interaction of the "fusion segment" with the target lipid bilayer or for triggering the membrane fusion.
为阐明甲型流感病毒（H1N1）血凝素（HA）“融合片段”中酸性氨基酸残基在pH依赖的膜融合中的作用，我们通过SV40 - HA病毒载体（SVHA）在CV - 1细胞中构建并表达了五个突变HA cDNA。通过脂质混合和多核体形成试验检测了这五个突变HA的融合活性。尽管用甘氨酸和赖氨酸取代了酸性残基，但通过脂质混合试验发现所有突变体均保留了其低pH依赖的融合活性。虽然SVHA - G19（HA(2)19D→G）、-K11（HA(2)11E→K）和-K19（HA(2)19D→K）与野生型HA一样在低pH下诱导多核体形成，但SVHA - G11（HA(2)11E→G）诱导的多核体形成有限，而SVHA - G11,19（HA(2)11E→G，19D→G）则未诱导多核体形成。在第11位用甘氨酸取代谷氨酸抑制了初始融合孔的扩大。然而，赖氨酸突变体在低pH下诱导形成初始融合孔并使其扩大，此时赖氨酸残基可能带正电荷。这些结果表明，在低pH下“融合片段”中酸性残基电荷的中和对于“融合片段”与靶脂质双层的相互作用或触发膜融合并不重要。
