RESUMEN

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.

Asunto(s)

Antígenos de la Hepatitis B/metabolismo , Virus de la Hepatitis B/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo D , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Transactivadores/metabolismo , Sitios de Unión , Unión Competitiva , Cromatografía de Afinidad , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Enteropeptidasa/metabolismo , Escherichia coli , Expresión Génica , Vectores Genéticos , Antígenos de la Hepatitis B/genética , Antígenos de la Hepatitis B/aislamiento & purificación , Virus de la Hepatitis B/genética , Hepatitis B Crónica/epidemiología , Ribonucleoproteína Nuclear Heterogénea D0 , Reacción en Cadena de la Polimerasa , Unión Proteica , Sondas ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Transactivadores/genética , Transactivadores/aislamiento & purificación , Proteínas Reguladoras y Accesorias Virales

Asunto(s)

Diarrea Infantil/enzimología , Galactosidasas/metabolismo , Yeyuno/enzimología , Sacarasa/metabolismo , alfa-Glucosidasas/metabolismo , beta-Galactosidasa/metabolismo , Enfermedad Crónica , Enteropeptidasa/metabolismo , Humanos , Lactante , Yeyuno/patología