An epitope of the Semliki Forest virus fusion protein exposed during virus-membrane fusion
An epitope of the Semliki Forest virus fusion protein exposed during virus-membrane fusion
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DOI：
10.1128/jvi.73.12.10029-10039.1999
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发表时间：
1999-12-01
影响因子：
5.4
通讯作者：
Kielian, M
中科院分区：
文献类型：
--
作者：
Ahn, A;Klimjack, MR;Kielian, M
塞姆利基森林病毒（SFV）是一种有包膜甲病毒，通过内吞途径中酸性pH触发的膜融合反应感染细胞，融合由刺突蛋白El亚基介导，该亚基是一种整合膜蛋白，含有病毒融合肽并在融合过程中形成稳定的同源三聚体。我们已经表征了四种特异于El酸性构象的单克隆抗体（MAb），这些MAb不抑制融合，表明它们与不同于融合肽的E1区域结合。竞争分析表明，所有四种 MAb 均与酸处理的病毒粒子或分离的刺突蛋白上的空间相关位点结合。为了绘制结合位点图谱，我们选择了对其中一种单克隆抗体 Ela-1 具有抗性的病毒突变体，一种病毒分离株 SFV 4-2 显示出包括 Ela-1 在内的三种酸特异性单克隆抗体的结合减少，而一种酸特异性单克隆抗体以及非酸特异性单克隆抗体与 E1 和 E2 的结合则没有变化。 SFV 4-2突变体具有完全感染性，形成F1同源三聚体，并具有野生型感染的pH依赖性。序列分析表明，SFV 4-2 中的相关突变是 E1 甘氨酸 157 变为精氨酸 (G157R)。在多种测定条件下观察到 MAb Ela-1 的结合减少，强烈表明 E1 G157R 突变直接影响 MAb 结合位点。因此，这些数据定位了通常隐藏在中性 pH 结构中的 E1 区域，并作为刺突蛋白重组为其融合活性构象的一部分而暴露出来。
Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by acidic pH in the endocytic pathway, Fusion is mediated by the spike protein El subunit, an integral membrane protein that contains the viral fusion peptide and forms a stable homotrimer during fusion, We have characterized four monoclonal antibodies (MAbs) specific for the acid conformation of El, These MAbs did not inhibit fusion, suggesting that they bind to an E1 region different from the fusion peptide. Competition analyses demonstrated that all four MAbs bound to spatially related sites on acid-treated virions or isolated spike proteins. To map the binding site, we selected for virus mutants resistant to one of the MAbs, Ela-1, One virus isolate, SFV 4-2, showed reduced binding of three acid-specific MAbs including Ela-1, while its binding of one acid-specific MAb as well as non-acid-specific MAbs to E1 and E2 was unchanged. The SFV 4-2 mutant was fully infectious, formed the F1 homotrimer, and had the wild-type pH dependence of infection. Sequence analysis demonstrated that the relevant mutation in SFV 4-2 was a change of E1 glycine 157 to arginine (G157R). Decreased binding of MAb Ela-1 was observed under a wide range of assay conditions, strongly suggesting that the E1 G157R mutation directly affects the MAb binding site. These data thus localize an E1 region that is normally hidden in the neutral pH structure and becomes exposed as part of the reorganization of the spike protein to its fusion-active conformation.