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Journal of Virology, December 1999, p. 10540-10545, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Characterization of the Phosphorylated Forms and the Phosphorylated Residues of Hepatitis Delta Virus Delta Antigens

Jung-Jung Mu,1 Hui-Lin Wu,2 Bor-Luen Chiang,3 Ruo-Ping Chang,3 Ding-Shinn Chen,2 and Pei-Jer Chen1,2,3,*

Graduate Institute of Microbiology,1 Hepatitis Research Center,2 and Graduate Institute of Clinical Medicine,3 College of Medicine, National Taiwan University, Taipei, Taiwan

Received 26 May 1999/Accepted 23 August 1999

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 32P 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.

* Corresponding author. Mailing address: Graduate Institute of Clinical Medicine, National Taiwan University Hospital, No. 7 Chung-Shan South Rd., Taipei, Taiwan. Phone: 886-2-23970800, ext. 7072. Fax: 886-2-23317624. E-mail: peijer{at}ha.mc.ntu.edu.tw.

Journal of Virology, December 1999, p. 10540-10545, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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