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 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.
Rui
E
E
Centro de Biologia Molecular Estrutural (CBME), Laboratório Nacional de Luz Síncrotron (LNLS), Rua Giuseppe Máximo Scolfaro 10.000, , Campinas-SP, Brazil.
de Moura
P R
PR
Kobarg
J
J
D001665:Binding Sites; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000378:metabolism; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors; D006511:Hepatitis B Antigens / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006515:Hepatitis B virus / Q000235:genetics / Q000378:metabolism*; D019694:Hepatitis B, Chronic / Q000453:epidemiology; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D015347:RNA Probes / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D006511:Hepatitis B Antigens; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D015347:RNA Probes; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D012313:RNA; D004765:Enteropeptidase