ISOLATION AND MOLECULAR CHARACTERIZATION OF HEPATITIS-B VIRUS X-PROTEIN FROM A BACULOVIRUS EXPRESSION SYSTEM

The X protein (HBx) of the human Hepatitis B Virus (HBV) is a regulato ry protein that exercises a transcriptional activator function on a va riety of regulatory elements and is therefore considered to be involve d in the development of human hepatocellular carcinoma (HCC). So far, most attempts at elucidating HBx function have been undertaken at the genetic level, reflecting the difficulties in detecting the very low a mounts of the protein in infected livers. Consequently, the questions of intracellular localization and posttranslational modification have not yet been completely answered, We therefore constructed recombinant baculoviruses that allowed expression of HBx and the hexa histidine H Bx fusion protein HBx(His) in insect cells. Cell fractionation experim ents revealed that only a minor part of HBx is detectable in a soluble form in the cytosolic fraction, whereas most of the protein forms int racellular aggregates. These results could be confirmed by confocal la ser immunofluorescence. The fusion of a hexa-histidine tag to the amin o terminus of HBx allowed a rapid one-step purification by metal chela te affinity chromatography. The detailed analysis of purified HBx(His) using electrospray ionization mass spectrometry uncovered two major c omponents: the unmodified, monomeric, fully oxidized form with five in tramolecular disulfide bridges, and its N-acetylated modification. Add itionally, two minor peaks with mass differences of Delta m = +80 da s uggested that a small fraction of HBx becomes posttranslationally phos phorylated in insect cells. No further modifications could be observed , indicating that only phosphorylation might play a role in a possible posttranslational regulation of this viral activator.