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. (C) 2004 Elsevier B.V. All rights reserved.10841671121131