Characterization of a hepatitis B and hepatitis delta virus receptor binding site
Characterization of a hepatitis B and hepatitis delta virus receptor binding site
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DOI：
10.1002/hep.21112
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发表时间：
2006-04-01
期刊：
影响因子：
13.5
通讯作者：
Urban, S
中科院分区：
文献类型：
--
作者：
Engelke, M;Mills, K;Urban, S
由于缺乏支持这些重要病原体完整复制周期的细胞培养系统，对人类乙型肝炎B（HBV）和丁型肝炎病毒（HDV）早期感染事件的了解受到限制。人肝癌细胞系HepaRG允许分化状态的实验诱导，从而获得对HBV和HDV感染的易感性。我们最近鉴定了HBV包膜蛋白衍生的脂肽，其包含L-表面蛋白的前S结构域的氨基酸2至48，其在皮摩尔浓度下已经阻断感染。为了绘制肽活性的负责序列，我们描述了一种允许豆蔻酰化的大肠杆菌表达系统，并研究了具有缺失突变和点突变的重组HBVpreS-GST融合蛋白预防HBV和HDV感染的能力。我们发现（1）肉豆蔻酰化的HBVpreS/2-48-GST融合蛋白有效地干扰了HBV感染HepaRG细胞，（2）高度保守的preS 1序列中第11 - 21位氨基酸的缺失和点突变导致感染抑制活性的丧失，（3）携带该区域内的单个氨基酸交换的B肝炎病毒失去感染性;（4）豆蔻酰化HBVpreS肽对HDV感染HepaRG细胞有同样的特异性抑制作用。总之，HBV和HDV使用至少一个共同的步骤进入肝细胞，并且需要L蛋白内高度保守的preS 1序列。该步骤对酰化HBVpreS 1肽的失活异常敏感，因此酰化HBVpreS 1肽代表了一组新的进入抑制剂，可用于治疗B型和D型肝炎。
Insights into the early infection events of the human hepatitis B (HBV) and hepatitis delta virus (HDV) have been limited because of the lack of a cell culture system supporting the full replication cycle for these important pathogens. The human hepatoma cell line HepaRG allows the experimental induction of a differentiated state, thereby gaining susceptibility toward HBV and HDV infection. We recently identified HBV envelope protein-derived lipopeptides comprising amino acids 2 though 48 of the preS-domain of the L-surface protein, which block infection already at picomolar concentrations. To map the responsible sequence for the peptides' activity we describe an Escherichia coli expression system that permits myristoylation and investigated recombinant HBVpreS-GST fusion proteins with deletion- and point-mutations for their ability to prevent HBV and HDV infection. We found that (1) a myristoylated HBVpreS/2-48-GST fusion protein efficiently interferes with HBV infection of HepaRG cells; (2) deletions and point mutations in the highly conserved preS1 sequence between amino acids 11 through 21 result in the loss of infection inhibition activity, (3) hepatitis B viruses carrying single amino acid exchanges within this region lose infectivity; and (4) HDV infection of HepaRG cells can be inhibited by myristoylated HBVpreS peptides with the same specificity. In conclusion, HBV and HDV use at least one common step to enter hepatocytes and require a highly conserved preS1-sequence within the L-protein. This step is exceptionally sensitive toward inactivation by acylated HBVpreS1 peptides, which therefore represent a novel group of entry inhibitors that could be used for the treatment of hepatitis B and D.