Role of the simian virus 5 fusion protein N-terminal coiled-coil domain in folding and promotion of membrane fusion
Role of the simian virus 5 fusion protein N-terminal coiled-coil domain in folding and promotion of membrane fusion
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DOI：
10.1128/jvi.79.3.1543-1551.2005
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发表时间：
2005-02-01
影响因子：
5.4
通讯作者：
Dutch, RE
中科院分区：
文献类型：
--
作者：
West, DS;Sheehan, MS;Dutch, RE
在 N 端卷曲螺旋的凹槽中形成由三个 C 端七肽重复区域以反平行方向组成的六螺旋束对于副粘病毒融合 (F) 蛋白促进膜融合至关重要。我们研究了猿猴病毒 5 (SV5) F 蛋白 N 端七肽重复序列的四个残基的突变对蛋白折叠、转运和融合活性的影响。先前对分离肽的研究表明，所选择的残基对 N 端卷曲螺旋和六螺旋束的稳定性具有不同的影响（R.E.Dutch、G.P.Leser 和 R.A.Lamb，Virology 254：147-159，1999）。突变体 V154M 显示蛋白水解裂解和表面表达减少，表明细胞内运输存在缺陷，尽管在分离的肽中研究时该突变没有影响。先前显示突变 I137M 会降低六螺旋束的热稳定性，产生 F 蛋白，该蛋白经过适当加工并转运至细胞表面，但融合活性降低。最后，L140M 和 L161M 的突变（之前已显示会破坏分离的 N-1 肽的 α 螺旋形成，但不会影响六螺旋束的形成）产生了经过正确加工的 F 蛋白。有趣的是，与野生型 F 蛋白相比，L161M 突变体显示出合胞体形成增加并在较低温度下促进融合。这些结果表明，与 N 端卷曲螺旋或六螺旋束的形成分开的相互作用对于 SV5 F 蛋白的初始折叠和运输非常重要，并且破坏 N 端卷曲螺旋稳定性的突变可能会导致膜融合的刺激。
Formation of a six-helix bundle comprised of three C-terminal heptad repeat regions in antiparallel orientation in the grooves of an N-terminal coiled-coil is critical for promotion of membrane fusion by paramyxovirus fusion (F) proteins. We have examined the effect of mutations in four residues of the N-terminal heptad repeat in the simian virus 5 (SV5) F protein on protein folding, transport, and fusogenic activity. The residues chosen have previously been shown from study of isolated peptides to have differing effects on stability of the N-terminal coiled-coil and six-helix bundle (R. E. Dutch, G. P. Leser, and R. A. Lamb, Virology 254:147-159, 1999). The mutant V154M showed reduced proteolytic cleavage and surface expression, indicating a defect in intracellular transport, though this mutation had no effect when studied in isolated peptides. The mutation I137M, previously shown to lower thermostability of the six-helix bundle, resulted in an F protein which was properly processed and transported to the cell surface but which had reduced fusogenic activity. Finally, mutations at L140M and L161M, previously shown to disrupt alpha-helix formation of isolated N-1 peptides but not to affect six-helix bundle formation, resulted in F proteins that were properly processed. Interestingly, the L161M mutant showed increased syncytium formation and promoted fusion at lower temperatures than the wild-type F protein. These results indicate that interactions separate from formation of an N-terminal coiled-coil or six-helix bundle are important in the initial folding and transport of the SV5 F protein and that mutations that destabilize the N-terminal coiled-coil can result in stimulation of membrane fusion.