Identification of an N-terminal trimeric coiled-coil core within arenavirus glycoprotein 2 permits assignment to class I viral fusion proteins
Identification of an N-terminal trimeric coiled-coil core within arenavirus glycoprotein 2 permits assignment to class I viral fusion proteins
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DOI：
10.1128/jvi.00008-06
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发表时间：
2006-06-01
影响因子：
5.4
通讯作者：
Hengartner, Hans
中科院分区：
文献类型：
--
作者：
Eschli, Bruno;Quirin, Katharina;Hengartner, Hans
淋巴细胞性脉络丛脑膜炎病毒（LCMV）糖蛋白（GP）由跨膜亚基GP-2和受体结合亚基GP-1组成。两者合成为一个前体蛋白，并在切割后保持非共价连接。在这项研究中，我们确定了LCMV GP的低聚状态，并以适合结构分析的两种不同构象表达了它。对GP-2进行序列分析，鉴定出含有n端α -螺旋的三聚七聚体重复序列。凝胶过滤层析和动态光散射结果表明，与该区域匹配的α -螺旋肽形成了稳定的低聚物。相比之下，第二个α -螺旋肽对应于GP-2中预测的c端a-螺旋没有寡聚。完整GP-2外结构域的重新折叠显示三聚体全α复合物可能代表六螺旋束状态，这被认为是I类病毒融合蛋白的标志。基于这些结果，我们生成了一个由完整不可切割的LCMV GP外结构域c端融合到纤维蛋白的三聚基序组成的结构体。对分泌的融合蛋白进行凝胶过滤分析，鉴定出两个类似于230和440 kDa的复合物。这两种复合物都与一组构象抗体和线性抗体结合。交联证实这个230 kda的复合物是一个三聚体。440-kDa的配合物被发现代表二硫连接的三聚体对，因为部分还原将它们转化为250 kDa迁移的复杂物种。在电子显微镜下，230-kDa配合物表现为单个球形颗粒，没有玫瑰花结形成的迹象。我们的结果清楚地表明，沙粒病毒GP是一个三聚体，必须被认为是一类病毒融合蛋白家族的成员。
The lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) consists of the transmembrane subunit GP-2 and the receptor binding subunit GP-1. Both are synthesized as one precursor protein and stay noncovalently attached after cleavage. In this study, we determined the oligomeric state of the LCMV GP and expressed it in two different conformations suitable for structural analysis. Sequence analysis of GP-2 identified a trimeric heptad repeat pattern containing an N-terminal alpha-helix. An alpha-helical peptide matching this region formed a stable oligomer as revealed by gel filtration chromatography and dynamic light scattering. In contrast, a second alpha-helical peptide corresponding to a predicted C-terminal a-helix within GP-2 did not oligomerize. Refolding of the complete GP-2 ectodomain revealed trimeric all-alpha complexes probably representing the six-helix bundle state that is considered a hallmark of class I viral fusion proteins. Based on these results, we generated a construct consisting of the complete uncleavable LCMV GP ectodomain fused C-terminally to the trimeric motif of fibritin. Gel filtration analysis of the secreted fusion protein identified two complexes of similar to 230 and similar to 440 kDa. Both complexes bound to a set of conformational and linear antibodies. Cross-linking confirmed the 230-kDa complex to be a trimer. The 440-kDa complexes were found to represent disulfide-linked pairs of trimers, since partial reduction converted them to a complex species migrating at 250 kDa. By electron microscopy, the 230-kDa complexes appeared as single spherical particles and showed no signs of rosette formation. Our results clearly demonstrate that the arenavirus GP is a trimer and must be considered a member of the class I viral fusion protein family.