Characterization of the phosphorylated forms and the phosphorylated residues of hepatitis delta virus delta antigens.
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.
Mu
J J
JJ
Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Wu
H L
HL
Chiang
B L
BL
Chang
R P
RP
Chen
D S
DS
Chen
P J
PJ
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
D018963:Hepatitis Antigens; D035923:Hepatitis delta Antigens; D020033:Protein Isoforms; C442244:hepatitis delta virus large antigen; D013912:Threonine; D012694:Serine