A strategy for the identification of HBV receptors with HBV pseudotype

乙肝病毒的发现已有近半个世纪的历史。尽管有一些关于感染系统和受体分子的报道，但关于乙肝病毒受体的冰山还没有被阐明。因此，到目前为止，我们还没有找到一个真正的乙肝病毒受体，也没有一个有用的、方便的体内外感染机制，这使得我们无法了解准确的乙肝病毒生命周期和与之相关的疾病。在这里，我们设计并试图产生一种乙肝病毒假型，它具有一个含有逆转录病毒衣壳的病毒颗粒和一个被乙肝病毒膜蛋白包围的基因组。我们通过抗HBVS抗体的免疫沉淀和CsC l密度梯度超速离心法成功地产生了这种假型，随后分别针对逆转录病毒基因和GFP基因进行了RT-PCR。虽然我们建立的系统是建立在逆转录病毒基因组的生长依赖整合上的，因此在原代人肝细胞培养系统中很难观察到它的感染情况，但成功地产生乙肝病毒伪型将使我们有可能进行基于传染性的生物学实验来克隆乙肝病毒受体，并将有助于其分离和鉴定。我们还没有鉴定出候选的乙肝病毒受体。然而，有人认为，在来自肝细胞癌的普通培养细胞系中可能存在一种附着因子。

It is near a half century since hepatitis B virus(HBV) was identified. An iceberg of HBV receptor has not been elucidated, though there are some reports on infection systems and on the receptor molecules. Thus, we still have not reached finding a real HBV receptor and there have been no useful and convenient infection system in vitro and in vivo for HBV, which makes it impossible for us to understand a precise HBV life cycle and HBV involved related diseases. Here, we designed and tried to generate an HBV pseudotype, which had a viral particle containing a retrovirus capsid and a genome inside surrounded by HBV membrane proteins. We proved successful generation of this pseudotype by immunoprecipitation with anti-HBVs antibodies and by CsCl density gradient ultracentrifugation, followed by RT-PCR targeting a retroviral gene, a GFP gene in this case, respectively. Though our established system is constructed on growth dependent integration of retroviral genomes and thus was very hard to observe its infection in a primary human hepatocytes culture system, successful generation of the HBV pseudotype will make it possible for us to perform a biological assay to clone an HBV receptor based on infectivity and will facilitate its separation and identification.We have not identified an HBV receptor candidate yet. It has been suggested, however, that there could be an attachment factor in ordinary cultured cell lines derived from hepatocellular carcinomas.