Mutations in the Fusion Peptide and Adjacent Heptad Repeat Inhibit Folding or Activity of the Newcastle Disease Virus Fusion Protein

Paramyxovirus fusion proteins have two heptad repeat domains, HR1 and HR2, which have been implicated in the fusion activity of the protein. Peptides with sequences from these two domains form a six-stranded coiled coil, with the HR1 sequences forming a central trimer (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309–319, 1999; X. Zhao, M. Singh, V. N. Malashkevich, and P. S. Kim, Proc. Natl. Acad. Sci. USA 97:14172–14177, 2000). We have extended our previous mutational analysis of the HR1 domain of the Newcastle disease virus fusion protein, focusing on the role of the amino acids forming the hydrophobic core of the trimer, amino acids in the “a” and “d” positions of the helix from amino acids 123 to 182. Both conservative and nonconservative point mutations were characterized for their effects on synthesis, stability, proteolytic cleavage, and surface expression. Mutant proteins expressed on the cell surface were characterized for fusion activity by measuring syncytium formation, content mixing, and lipid mixing. We found that all mutations in the “a” position interfered with proteolytic cleavage and surface expression of the protein, implicating the HR1 domain in the folding of the F protein. However, mutation of five of seven “d” position residues had little or no effect on surface expression but, with one exception at residue 175, did interfere to various extents with the fusion activity of the protein. One of these “d” mutations, at position 154, interfered with proteolytic cleavage, while the rest of the mutants were cleaved normally. That most “d” position residues do affect fusion activity argues that a stable HR1 trimer is required for formation of the six-stranded coiled coil and, therefore, optimal fusion activity. That most of the “d” position mutations do not block folding suggests that formation of the core trimer may not be required for folding of the prefusion form of the protein. We also found that mutations within the fusion peptide, at residue 128, can interfere with folding of the protein, implicating this region in folding of the molecule. No characterized mutation enhanced fusion.

副黏病毒（Paramyxovirus）融合蛋白拥有两个七肽重复结构域HR1（heptad repeat 1）与HR2（heptad repeat 2），二者均与该蛋白的融合活性密切相关。源自这两个结构域序列的肽段可形成六股卷曲螺旋，其中HR1序列构成中心三聚体（K. A. Baker、R. E. Dutch、R. A. Lamb及T. S. Jardetzky，《分子细胞》，3卷：309–319，1999年；X. Zhao、M. Singh、V. N. Malashkevich及P. S. Kim，《美国国家科学院院刊》，97卷：14172–14177，2000年）。本研究拓展了此前针对新城疫病毒融合蛋白HR1结构域的突变分析工作，重点关注构成该三聚体疏水核心的氨基酸残基——即123至182位氨基酸所在螺旋的“a”位与“d”位残基。我们对保守点突变与非保守点突变分别进行了表征，以探究其对蛋白合成、稳定性、蛋白水解切割及细胞表面表达的影响。针对表达于细胞表面的突变蛋白，我们通过检测合胞体形成、内容混合与脂质混合水平，对其融合活性进行了表征。研究发现，所有“a”位残基的突变均会干扰该蛋白的蛋白水解切割与细胞表面表达，这表明HR1结构域参与F蛋白（fusion protein）的折叠过程。然而，7个“d”位残基中的5个发生突变后，对细胞表面表达几乎无影响，但除175位残基的突变外，其余均在不同程度上干扰了该蛋白的融合活性。其中154位“d”位突变会干扰蛋白水解切割，其余突变体的蛋白水解过程均正常。多数“d”位残基突变会影响融合活性这一结果表明，稳定的HR1三聚体是形成六股卷曲螺旋的必要条件，亦是实现最优融合活性的前提。多数“d”位突变并未阻断蛋白折叠，这提示核心三聚体的形成或许并非蛋白预融合构象折叠所必需的步骤。我们还发现，融合肽段（fusion peptide）内128位残基的突变可干扰蛋白折叠，表明该区域参与了蛋白分子的折叠过程。本研究未发现任何经过表征的突变可增强蛋白的融合活性。