Rui E, de Moura P R, Kobarg J
Centro de Biologia Molecular Estrutural (CBME), Laboratório Nacional de Luz Síncrotron (LNLS), Rua Giuseppe Máximo Scolfaro 10.000, , Campinas-SP, Brazil.
Virus Res. 2001 Apr;74(1-2):59-73. doi: 10.1016/s0168-1702(00)00245-8.
The hepatitis B virus protein HBx has been implicated in the development of liver cancer. It has been shown that the HBx protein is able to bind to single-stranded DNA in a specific manner. This DNA binding activity might be relevant for HBx oncogene character. To study the HBx interaction with nucleic acids in more detail we expressed full-length HBx as well as several N- and C-terminally truncated HBx proteins as 6xHis and GST-fusions in E. coli. Using a gel shift assay, we were able to demonstrate that all of the truncated HBx proteins have the ability to bind to an AU-rich RNA. The affinity of GST-HBx #3 (residues 80-142) was an order of magnitude higher than that of GST-HBx #2 (residues 5-79), indicating that a high affinity RNA binding site is located in HBx C-terminal half. AUF1 is the protein ligand that binds to AU-rich RNA regions present in certain proto-oncogene mRNAs and causes their rapid degradation. By a competitive binding experiment of AUF1 and HBx to the AU-rich RNA oligonucleotide, we show that HBx is able to displace AUF1 from its binding site on the RNA oligonucleotide. This new aspect of HBx function is discussed in the context of cellular transformation.
乙肝病毒蛋白HBx与肝癌的发生发展有关。研究表明，HBx蛋白能够以特定方式与单链DNA结合。这种DNA结合活性可能与HBx癌基因特性相关。为了更详细地研究HBx与核酸的相互作用，我们在大肠杆菌中表达了全长HBx以及几种N端和C端截短的HBx蛋白，它们分别作为6xHis融合蛋白和GST融合蛋白。通过凝胶迁移实验，我们能够证明所有截短的HBx蛋白都有能力结合富含AU的RNA。GST-HBx #3（第80 - 142位氨基酸残基）的亲和力比GST-HBx #2（第5 - 79位氨基酸残基）高一个数量级，这表明高亲和力RNA结合位点位于HBx的C端一半区域。AUF1是一种蛋白质配体，它能与某些原癌基因mRNA中存在的富含AU的RNA区域结合，并导致其快速降解。通过AUF1和HBx与富含AU的RNA寡核苷酸的竞争性结合实验，我们发现HBx能够将AUF1从其在RNA寡核苷酸上的结合位点置换下来。本文在细胞转化的背景下讨论了HBx功能的这一新方面。
