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Detection of the human hepatitis B virus X-protein in transgenic mice after radioactive labelling at a newly introduced phosphorylation site

Department of Virology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany¹

Author for correspondence: Eva Schmitteckert. Present address: Institute for Pharmacology and Toxicology, University of Wuerzburg, Versbacher Str. 9, D-97078 Wuerzburg, Germany. Fax +49 931 201 3539. e-mail schmitteckert{at}toxi.uni-wuerzburg.de

Besides the three essential genes encoding the envelope, core and polymerase proteins, all mammalian hepadnaviruses examined to date contain a fourth gene which is referred to as the x-gene. This gene is believed to encode a transcriptional transactivator which positively regulates viral gene expression. Attempts to detect X-protein in vivo or in tissue culture lead to varying results. Whereas some groups could detect a protein of the expected size, other groups did not. To establish optimal conditions for the isolation of the human hepatitis B virus X-protein, we introduced a recognition site for protein kinase A into the x-gene. Upon phosphorylation with radioactive ATP, this modified X-protein can be detected with very high specificity and sensitivity. Tissue culture experiments showed that X-protein expressed from a cytomegalovirus-driven plasmid is not soluble in non-ionic detergent but rather has to be extracted from the cell pellet by boiling with SDS at a slightly alkaline pH. This method was then used to examine the organs of several transgenic mouse lines which expressed the modified x-gene under control of the authentic promoter. The data show that expression of the x-gene and subsequent biosynthesis of the X-protein is not tissue-specific but rather can occur in most organs.