The cysteine residues of the hepatitis B virus onco-protein HBx are not required for its interaction with RNA or with human p53.
The hepatitis B virus (HBV) protein HBx has been implicated to induce liver cancer in transgenic mice and transactivates a variety of viral and cellular promoters. The 17 kDa protein HBx consists of 154 amino acids, contains 10 cysteine residues and is translated during the viral infection. It has been shown previously that the HBx protein is able to bind to singlestranded DNA and RNA. This nucleic acid binding activity might be relevant for HBx oncogenic character. Furthermore, HBx has been reported to interact with a series of cellular proteins, especially with transcription factors, including the tumor suppressor protein p53. To evaluate the importance of the cysteine residues in HBx for its interaction with RNA and p53 we expressed full-length HBx-wt as well as several truncated mini-HBx(18-142) proteins with multiple cysteine to serine point mutations as 6xHis fusion proteins in Escherichia coli. Using UV cross-linking assays we demonstrate that all truncated mini-HBx proteins with cysteine/serine point mutations maintained the ability to bind to an AU-38 RNA oligonucleotide. Furthermore, we performed in vitro binding assays of selected HBx mutants with GST-p53, circular dichroism spectroscopic analysis of the mutant HBx protein secondary structure and a p53 based transcription activation assay in yeast cells. In summary, our data suggest that the cysteine residues in the HBx protein are of minor importance for its interaction with both RNA and the p53 protein.
de Moura
Patricia Ribeiro
PR
Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, Rua Giuseppe Máximo Scolfaro 10.000, CP 6192, Campinas, SP, CEP 13084-971, Brazil.
Rui
Edmilson
E
de Almeida Gonçalves
Kaliandra
K
Kobarg
Jörg
J
D019943:Amino Acid Substitution; D002942:Circular Dichroism; D003545:Cysteine / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006515:Hepatitis B virus / Q000502:physiology*; D006801:Humans; D020125:Mutation, Missense; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D011994:Recombinant Proteins; D015534:Trans-Activators; D016159:Tumor Suppressor Protein p53; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D012313:RNA; D003545:Cysteine