Mu J.-J.Wu H.-L.BOR-LUEN CHIANGChang R.-P.DING-SHINN CHENPEI-JER CHEN2021-07-022021-07-0219990022-538Xhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-84983726735&doi=10.1128%2fjvi.73.12.10540-10545.1999&partnerID=40&md5=c52d134414994fde10034d8d1c9eeb3ahttps://scholars.lib.ntu.edu.tw/handle/123456789/568103Hepatitis 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 vital 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.[SDGs]SDG3hepatitis delta antigen; phorbol 13 acetate 12 myristate; phosphoprotein; article; Hepatitis delta virus; nonhuman; priority journal; protein phosphorylation; two dimensional gel electrophoresisjournal article10.1128/jvi.73.12.10540-10545.1999105593752-s2.0-84983726735