title: Characterization of the phosphorylated forms and the phosphorylated residues of hepatitis delta virus delta antigens.
abstract: Hepatitis delta virus (HDV) replication requires both the cellular RNA polymerase and one virus-encoded protein, small delta antigen (S-HDAg). S-HDAg has been shown to be a phosphoprotein, but its phosphorylation status is not yet clear. In this study, we employed three methods to address this question. A special two-dimensional gel electrophoresis, namely, nonequilibrium pH gradient electrophoresis, was used to separate the very basic S-HDAg. By carefully adjusting the pH of solubilization solution, the ampholyte composition, and the appropriate electrophoresis time periods, we were able to clearly resolve S-HDAg into two phosphorylated isoforms and one unphosphorylated form. In contrast, the viral large delta antigen (L-HDAg) can only be separated into one phosphorylated and one unphosphorylated form. By metabolic (32)P labeling, both immunoprecipitated S-HDAg and L-HDAg were found to incorporate radioactive phosphate. The extent of S-HDAg phosphorylation was increased upon 12-O-tetradecanoylphorbol-13-acetate treatment, while that of L-HDAg was not affected. Finally, phosphoamino acid analysis identified serine and threonine as the phospho residues in the labeled S-HDAg and only serine in the L-HDAg. Therefore, HDV S- and L-HDAgs differ in their phosphorylation patterns, which may account for their distinct biological functions.
authors.affiliation: Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: J J
authors.initials: JJ
authors.lastname: Mu
authors.forename: H L
authors.initials: HL
authors.lastname: Wu
authors.forename: B L
authors.initials: BL
authors.lastname: Chiang
authors.forename: R P
authors.initials: RP
authors.lastname: Chang
authors.forename: D S
authors.initials: DS
authors.lastname: Chen
authors.forename: P J
authors.initials: PJ
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D018963:Hepatitis Antigens / Q000378:metabolism*; D003698:Hepatitis Delta Virus; D035923:Hepatitis delta Antigens; D006801:Humans; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D010766:Phosphorylation; D020033:Protein Isoforms; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D018963:Hepatitis Antigens; D035923:Hepatitis delta Antigens; D020033:Protein Isoforms; C442244:hepatitis delta virus large antigen; D013912:Threonine; D012694:Serine