STUDIES ON THE MECHANISM OF MEMBRANE-FUSION - SITE-SPECIFIC MUTAGENESIS OF THE HEMAGGLUTININ OF INFLUENZA-VIRUS
STUDIES ON THE MECHANISM OF MEMBRANE-FUSION - SITE-SPECIFIC MUTAGENESIS OF THE HEMAGGLUTININ OF INFLUENZA-VIRUS
复制标题
DOI：
10.1083/jcb.102.1.11
复制
发表时间：
1986-01-01
影响因子：
7.8
通讯作者：
WHITE, J
中科院分区：
文献类型：
--
作者：
GETHING, MJ;DOMS, RW;WHITE, J
编码流感病毒血凝素的cDNA的寡核苷酸定向突变已被用于在编码HA2亚基氨基末端的保守极性“融合肽”的序列中引入单碱基变化。该突变序列取代了SV40-HA重组病毒载体中的野生型基因，改变后的HA蛋白在猿猴细胞中表达。我们构建了三个突变体，在融合肽中引入了单一的非保守氨基酸变化，并观察到三种融合表型：在HA2的氨基端，谷氨酸取代了甘氨酸残基，消除了所有融合活性；谷氨酸取代HA2中4位甘氨酸残基提高了阈值pH，降低了融合效率；最后，通过用甘氨酸取代第11位的谷氨酸来延长疏水拉伸，产生了一个突变蛋白，该突变蛋白诱导红细胞与细胞融合，其效率和pH谱与野生型蛋白相同。然而，该突变体诱导多核细胞形成的能力被大大削弱。然而，所有突变蛋白都经历了ph依赖性构象变化并与脂质体结合。这些结果在ha诱导的膜融合机制方面进行了讨论。
Oligonucleotide-directed mutagenesis of a cDNA encoding the hemagglutinin of influenza virus has been used to introduce single base changes into the sequence that codes for the conserved apolar "fusion peptide" at the amino-terminus of the HA2 subunit. The mutant sequences replaced the wild-type gene in SV40-HA recombinant virus vectors, and the altered HA proteins were expressed in simian cells. Three mutants have been constructed that introduce single nonconservative amino acid changes in the fusion peptide, and three fusion phenotypes were observed: substitution of glutamic acid for the glycine residue at the amino-terminus of HA2 abolished all fusion activity; substitution of glutamic acid for the glycine residue at position 4 in HA2 raised the threshold pH and decreased the efficiency of fusion; and, finally, extension of the hydrophobic stretch by replacement of the glutamic acid at position 11 with glycine yielded a mutant protein that induced fusion of erythrocytes with cells with the same efficiency and pH profile as the wild-type protein. However, the ability of this mutant to induce polykaryon formation was greatly impaired. Nevertheless, all the mutant proteins underwent a pH-dependent conformational change and bound to liposomes. These results are discussed in terms of the mechanism of HA-induced membrane fusion.