Gardner Amanda E, Martin Kimberly L, Dutch Rebecca E
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0509, USA.
Biochemistry. 2007 May 1;46(17):5094-105. doi: 10.1021/bi6025648. Epub 2007 Apr 7.
Paramyxoviruses are a diverse family that utilizes a fusion (F) protein to enter cells via fusion of the viral lipid bilayer with a target cell membrane. Although certain regions of the F protein are known to play critical roles in membrane fusion, the function of much of the protein remains unclear. Sequence alignment of a set of paramyxovirus F proteins and analysis utilizing Block Maker identified a region of conserved amino acid sequence in a large domain between the heptad repeats of F1, designated CBF1. We employed site-directed mutagenesis to analyze the function of completely conserved residues of CBF1 in both the simian virus 5 (SV5) and Hendra virus F proteins. The majority of CBF1 point mutants were deficient in homotrimer formation, proteolytic processing, and transport to the cell surface. For some SV5 F mutants, proteolytic cleavage and surface expression could be restored by expression at 30 degrees C, and varying levels of fusion promotion were observed at this temperature. In addition, the mutant SV5 F V402A displayed a hyperfusogenic phenotype at both 30 and 37 degrees C, indicating that this mutation allows for efficient fusion with only an extremely small amount of cleaved, active protein. The recently published prefusogenic structure of PIV5/SV5 F (Yin, H. S., et al. (2006) Nature 439, 38-44) indicates that residues within and flanking CBF1 interact with the fusion peptide domain. Together, these data suggest that CBF1-fusion peptide interactions are critical for the initial folding of paramyxovirus F proteins from this important viral family and can also modulate subsequent membrane fusion promotion.
副粘病毒是一个多样化的病毒家族，它利用融合（F）蛋白通过病毒脂质双层与靶细胞膜的融合进入细胞。虽然已知F蛋白的某些区域在膜融合中起关键作用，但该蛋白大部分区域的功能仍不清楚。一组副粘病毒F蛋白的序列比对以及利用Block Maker进行的分析确定了F1七肽重复序列之间一个大结构域中保守氨基酸序列的区域，命名为CBF1。我们采用定点诱变来分析CBF1在猿猴病毒5（SV5）和亨德拉病毒F蛋白中完全保守残基的功能。大多数CBF1点突变体在同源三聚体形成、蛋白水解加工以及转运到细胞表面方面存在缺陷。对于一些SV5 F突变体，在30℃表达时可恢复蛋白水解切割和表面表达，并且在此温度下观察到不同程度的融合促进作用。此外，突变体SV5 F V402A在30℃和37℃均表现出超融合表型，这表明该突变仅需极少量切割的活性蛋白就能实现高效融合。最近发表的PIV5/SV5 F的前融合结构（Yin, H. S.,等人（2006年）《自然》第439卷，38 - 44页）表明，CBF1内部及其侧翼的残基与融合肽结构域相互作用。总之，这些数据表明CBF1 - 融合肽相互作用对于这个重要病毒家族的副粘病毒F蛋白的初始折叠至关重要，并且还可以调节随后的膜融合促进作用。
