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

Besides the three essential genes encoding the envelope, core and polymeras e 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 expres sion. Attempts to detect X-protein in vivo or in tissue culture lead to var ying results. Whereas some groups could detect a protein of the expected si ze, 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 radioactiv e ATP, this modified X-protein can be detected with very high specificity a nd 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 c ontrol of the authentic promoter. The data show that expression of the x-ge ne and subsequent biosynthesis of the X-protein is not tissue-specific but rather can occur in most organs.