Reitter J N, Sergel T, Morrison T G
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester 01655, USA.
J Virol. 1995 Oct;69(10):5995-6004. doi: 10.1128/JVI.69.10.5995-6004.1995.
The paramyxovirus fusion proteins have a highly conserved leucine zipper motif immediately upstream from the transmembrane domain of the F1 subunit (R. Buckland and F. Wild, Nature [London] 338:547, 1989). To determine the role of the conserved leucines in the oligomeric structure and biological activity of the Newcastle disease virus (NDV) fusion protein, the heptadic leucines at amino acids 481, 488, and 495 were changed individually and in combination to an alanine residue. While single amino acid changes had little effect on fusion, substitution of two or three leucine residues abolished the fusogenic activity of the protein, although cell surface expression of the mutants was higher than that of the wild-type protein. Substitution of all three leucine residues with alanine did not alter the size of the fusion protein oligomer as determined by sedimentation in sucrose gradients. Furthermore, deletion of the C-terminal 91 amino acids, including the leucine zipper motif and transmembrane domain, resulted in secretion of an oligomeric polypeptide. These results indicate that the conserved leucines are not necessary for oligomer formation but are required for the fusogenic ability of the protein. When the polar face of the potential alpha helix was altered by nonconservative changes of serine to alanine (position 473), glutamic acid to lysine or alanine (position 482), asparagine to lysine (position 485), or aspartic acid to alanine (position 489), the fusogenic ability of the protein was not significantly disrupted. In addition, a double mutant (E482A,D489A) which removed negative charges along one side of the helix had negligible effects on fusion activity.
副粘病毒融合蛋白在F1亚基跨膜结构域上游紧邻处有一个高度保守的亮氨酸拉链基序（R. 巴克兰德和F. 怀尔德，《自然》[伦敦] 338:547，1989年）。为了确定保守亮氨酸在新城疫病毒（NDV）融合蛋白的寡聚结构和生物学活性中的作用，将481、488和495位氨基酸处的七聚体亮氨酸分别或组合替换为丙氨酸残基。虽然单个氨基酸变化对融合影响不大，但替换两个或三个亮氨酸残基会消除该蛋白的融合活性，尽管突变体在细胞表面的表达高于野生型蛋白。用丙氨酸替换所有三个亮氨酸残基，通过蔗糖梯度沉降测定，并未改变融合蛋白寡聚体的大小。此外，缺失包括亮氨酸拉链基序和跨膜结构域在内的C末端91个氨基酸，导致一种寡聚多肽的分泌。这些结果表明，保守亮氨酸对于寡聚体形成并非必需，但对于该蛋白的融合能力是必需的。当潜在α螺旋的极性面通过丝氨酸到丙氨酸（473位）、谷氨酸到赖氨酸或丙氨酸（482位）、天冬酰胺到赖氨酸（485位）或天冬氨酸到丙氨酸（489位）的非保守变化而改变时，该蛋白的融合能力并未受到显著破坏。此外，一个沿螺旋一侧去除负电荷的双突变体（E482A，D489A）对融合活性的影响可忽略不计。
