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 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.
Schmitteckert
Eva Maria
EM
Department of Virology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany1.
Schlicht
Hans-Jürgen
HJ
Department of Virology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany1.
D000818:Animals; D015153:Blotting, Western; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D009928:Organ Specificity; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012995:Solubility; D015534:Trans-Activators / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D054334:Viral Regulatory and Accessory Proteins
D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein