The disulfide bonds in glycoprotein E2 of hepatitis C virus reveal the tertiary organization of the molecule.
The disulfide bonds in glycoprotein E2 of hepatitis C virus reveal the tertiary organization of the molecule.
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DOI：
10.1371/journal.ppat.1000762
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发表时间：
2010-02-19
期刊：
影响因子：
6.7
通讯作者：
Rey FA
中科院分区：
文献类型：
--
作者：
Krey T;d'Alayer J;Kikuti CM;Saulnier A;Damier-Piolle L;Petitpas I;Johansson DX;Tawar RG;Baron B;Robert B;England P;Persson MA;Martin A;Rey FA
丙型肝炎病毒（HCV）是人类慢性肝病的主要原因，是全世界研究工作的重点。然而，尽管病毒包膜糖蛋白 E1 和 E2 在病毒生命周期中发挥着重要作用，但它们的结构数据却很少。为了获得更多信息，我们使用果蝇细胞开发了重组 E2 胞外域 (E2e) 的高效生产系统，该系统在其跨膜 (TM) 区域的上游被截短。该系统产生大部分单体蛋白质，可以通过色谱法轻松地将其与污染的二硫键连接的聚集体分离。分离的单体E2e与许多构象敏感的单克隆抗体反应，结合可溶性CD81大外环，并以剂量依赖性方式有效抑制感染性HCV颗粒（HCVcc）对Huh7.5细胞的感染，表明其采用天然构象。 E2e 的这些特性使我们通过实验确定了其 9 个二硫键的连接性，这些键在 HCV 基因型中严格保守。此外，圆二色性与红外光谱分析相结合揭示了E2e的二级结构含量，特别表明约28％的β-折叠，与一致的二级结构预测一致。二硫键连接模式与 CD81 结合位点和报道的 E2 缺失突变体的数据一起，使 E2e 多肽链能够连接到相关黄病毒和甲病毒的 II 类融合蛋白的结构模板上。由此产生的 E2 三级组织模型提供了有关病毒抗原性决定因素的关键信息，将受体结合位点映射到结构域 I 和 III 的界面，并提供了对推定融合构象变化本质的深入了解。尽管丙型肝炎病毒 (HCV) 的包膜糖蛋白在这种主要人类病原体的病毒周期中发挥着重要作用，但人们对它们的结构知之甚少。在这里，我们确定了 HCV 糖蛋白 E2 胞外域严格保守的 18 个半胱氨酸形成的 9 个二硫键的连接性。我们表明，这些信息与文献中可用的重要功能数据一起，对分子可能的三维折叠施加了重要限制。事实上，这些限制允许将预测的二级结构元件沿着多肽链明确地连接到由相关黄病毒和甲病毒II类融合蛋白的晶体结构提供的模板上。由此产生的 E2 三级组织模型显示了特征性 II 类结构域之间的氨基酸分布，将 CD81 结合位点置于结构域 I 和 III 的界面处，并突出显示了候选融合环的位置。
Hepatitis C virus (HCV), a major cause of chronic liver disease in humans, is the focus of intense research efforts worldwide. Yet structural data on the viral envelope glycoproteins E1 and E2 are scarce, in spite of their essential role in the viral life cycle. To obtain more information, we developed an efficient production system of recombinant E2 ectodomain (E2e), truncated immediately upstream its trans-membrane (TM) region, using Drosophila melanogaster cells. This system yields a majority of monomeric protein, which can be readily separated chromatographically from contaminating disulfide-linked aggregates. The isolated monomeric E2e reacts with a number of conformation-sensitive monoclonal antibodies, binds the soluble CD81 large external loop and efficiently inhibits infection of Huh7.5 cells by infectious HCV particles (HCVcc) in a dose-dependent manner, suggesting that it adopts a native conformation. These properties of E2e led us to experimentally determine the connectivity of its 9 disulfide bonds, which are strictly conserved across HCV genotypes. Furthermore, circular dichroism combined with infrared spectroscopy analyses revealed the secondary structure contents of E2e, indicating in particular about 28% β-sheet, in agreement with the consensus secondary structure predictions. The disulfide connectivity pattern, together with data on the CD81 binding site and reported E2 deletion mutants, enabled the threading of the E2e polypeptide chain onto the structural template of class II fusion proteins of related flavi- and alphaviruses. The resulting model of the tertiary organization of E2 gives key information on the antigenicity determinants of the virus, maps the receptor binding site to the interface of domains I and III, and provides insight into the nature of a putative fusogenic conformational change. Little is known about the structure of the envelope glycoproteins of the hepatitis C virus (HCV), in spite of their essential role in the viral cycle of this major human pathogen. Here, we determined the connectivity of the 9 disulfide bonds formed by the strictly conserved 18 cysteines of the ectodomain of HCV glycoprotein E2. We show that this information, together with important functional data available in the literature, impose important restrictions to the possible three-dimensional fold of the molecule. Indeed, these constraints allow the unambiguous threading of the predicted secondary structure elements along the polypeptide chain onto the template provided by the crystal structures of related flavi- and alphavirus class II fusion proteins. The resulting model of the tertiary organization of E2 shows the amino acid distribution among the characteristic class II domains, places the CD81 binding site at the interface of domains I and III, and highlights the location of a candidate fusion loop.
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发表时间：
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影响因子：
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