Expression of deletion mutants of the hepatitis B virus protein HBx in E-coli and characterization of their RNA binding activities

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 si ngle-stranded DNA in a specific manner. This DNA binding activity might be relevant for HBx oncogene character. To study the HBx interaction with nucl eic 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. col i. Using a gel shift assay, we were able to demonstrate that all of the tru ncated HBx proteins have the ability to bind to an AU-rich RNA. The affinit y of GST-HBx # 3 (residues 80-142) was an order of magnitude higher than th at of GST-HBx # 2 (residues 5-79), indicating that a high affinity RNA bind ing 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 c auses 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 dis place AUF1 from its binding site on the RNA oligonucleotide. This new aspec t of HBx function is discussed in the context of cellular transformation. ( C) 2001 Elsevier Science B.V. All rights reserved.