Functional properties of a monoclonal antibody inhibiting the hepatitis C virus RNA-dependent RNA polymerase
Functional properties of a monoclonal antibody inhibiting the hepatitis C virus RNA-dependent RNA polymerase
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DOI：
10.1074/jbc.m108748200
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发表时间：
2002-01-04
影响因子：
4.8
通讯作者：
Bartenschlager, R
中科院分区：
文献类型：
--
作者：
Moradpour, D;Bieck, E;Bartenschlager, R
以非结构蛋白 513 (NS5B) 为代表的丙型肝炎病毒 (HCV) RNA 依赖性 RNA 聚合酶 (RdRp) 最近已成为抗病毒干预的一个有前景的靶标。在这里，我们描述了抑制 HCV RdRp 的单克隆抗体 (mAb) 的分离、功能表征和分子克隆。该m)Ab，指定为5B-12B7，以高亲和力结合HCV RdRp掌子域中的构象表位，并识别在整个HCV多蛋白或亚基因组复制子的背景下表达的天然NS5B。在等摩尔浓度的 NS5B 和 mAb 5B-12B7 下观察到 RdRp 活性在体外完全抑制，而猪瘟病毒 NS5B 和脊髓灰质炎病毒 3D 聚合酶的 RdRp 活性不受影响。 mAb 5B-12B7 选择性抑制 NTP 与 HCV NS5B 的结合，而模板 RNA 的结合不受影响，从而在分子水平上解释了作用机制。通过逆转录PCR克隆mAb 5B-12B7重链和轻链可变结构域，并组装单链Fv片段以在大肠杆菌和真核细胞中表达。 mAb 5B-12B7 单链 Fv 片段在体外和转染的人细胞系中均与 NS5B 结合，因此可能可用于针对 HCV 的细胞内免疫。更重要的是，对酶上 mAb 5B-12B7 接触位点的详细了解可能会促进小分子 RdRp 抑制剂作为新型抗病毒药物的开发。
The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 513 (NS5B), has recently emerged as a promising target for antiviral intervention. Here, we describe the isolation, functional characterization, and molecular cloning of a monoclonal antibody (mAb) inhibiting the HCV RdRp. This m)Ab, designated 5B-12B7, binds with high affinity to a conformational epitope in the palm subdomain of the HCV RdRp and recognizes native NS5B expressed in the context of the entire HCV polyprotein or subgenomic replicons. Complete inhibition of RdRp activity in vitro was observed at equimolar concentrations of NS5B and mAb 5B-12B7, whereas RdRp activities of classical swine fever virus NS5B and poliovirus 3D polymerase were not affected. mAb 5B-12B7 selectively inhibited NTP binding to HCV NS5B, whereas binding of template RNA was unaffected, thus explaining the mechanism of action at the molecular level. The mAb 5B-12B7 heavy and light chain variable domains were cloned by reverse transcription-PCR, and a single chain Fv fragment was assembled for expression in Escherichia coli and in eukaryotic cells. The mAb 5B-12B7 single chain Fv fragment bound to NS5B both in vitro and in transfected human cell lines and therefore may be potentially useful for intracellular immunization against HCV. More important, detailed knowledge of the mAb 5B-12B7 contact sites on the enzyme may facilitate the development of small molecule RdRp inhibitors as novel antiviral agents.