Phosphorylation and rapid turnover of hepatitis B virus X-protein expressed in HepG2 cells from a recombinant vaccinia virus.
The human hepatitis B viral (HBV) genome contains a conserved open reading frame known as the X-gene which is capable of encoding a polypeptide of 16.565 kDa. The corresponding protein has so far not been identified directly in HBV-infected cells, but in transient transfection assays the X-gene encodes a product that functions as a transcriptional transactivator. To characterize the subcellular distribution, stability and post-translational modifications of X-protein in human hepatoma HepG2 cells, we have established a vaccinia virus expression system. As the major X-gene product, a protein with an apparent molecular weight of 16 kDa, and reacting with an X-protein-specific antiserum, was expressed from recombinant vaccinia virus. In indirect immunofluorescence assay, X-protein appeared to be distributed throughout the cells, with a tendency to localize at the nuclear periphery and to accumulate in granules as its levels increased. By subcellular fractionation, we found about one-third of X-protein associated with the fraction defined as the nuclear framework. In pulse-chase experiments, X-protein decayed with a bimodal half-life of 15 min and 3 h. X-protein having a half-life of about 15 min was found associated with the Triton X-100 detergent-soluble fraction of HepG2 cells, while that associated with the insoluble fraction turned over more slowly. By metabolic labeling with [32P] orthophosphate, we show that X-protein is capable of being phosphorylated. Modification by phosphorylation could play an important role in the regulation of X-protein function.
Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg, Germany.
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N
Schek
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R
Bartenschlager
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C
Kuhn
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H
Schaller
D000595:Amino Acid Sequence; D003062:Codon; D016679:Genome, Viral; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010766:Phosphorylation; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D003062:Codon; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein