Mu J.-J.Chen D.-S.PEI-JER CHEN2021-07-032021-07-0320010022-538Xhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-84983719356&doi=10.1128%2fJVI.75.19.9087-9095.2001&partnerID=40&md5=b41d10f0040574d7cf7b15f7a00b6f1ehttps://scholars.lib.ntu.edu.tw/handle/123456789/568797Hepatitis delta virus (HDV) small delta antigen (S-HDAg) plays a critical role in virus replication. We previously demonstrated that the S-HDAg phosphorylation occurs on both serine and threonine residues. However, their biological significance and the exact phosphorylation sites of S-HDAg are still unknown. In this study, phosphorylated S-HDAg was detected only in the intracellular compartment, not in viral particles. In addition, the number of phosphorylated isoforms of S-HDAg significantly increased with the extent of viral replication in transfection system. Site-directed mutagenesis showed that alanine replacement of serine 177, which is conserved among all the known HDV strains, resulted in reduced phosphorylation of S-HDAg, while the mutation of the other two conserved serine residues (2 and 123) had little effect. The S177A mutant dramatically decreased its capability in assisting HDV RNA replication, with a preferential and profound impairment of the antigenomic RNA replication. Furthermore, the viral RNA editing, a step relying upon antigenomic RNA replication, was also abolished by this mutation. These results suggested that phosphorylation of S-HDAg, with serine 177 as a presumable site, plays a critical role in viral RNA replication, especially in augmenting the replication of antigenomic RNA.[SDGs]SDG3virus RNA; amino acid substitution; article; DNA transfection; Hepatitis delta virus; immunoprecipitation; nonhuman; Northern blotting; phosphorylation; polyacrylamide gel electrophoresis; priority journal; reverse transcription polymerase chain reaction; RNA editing; RNA replication; site directed mutagenesis; virus mutation; Western blottingjournal article10.1128/JVI.75.19.9087-9095.2001115331722-s2.0-84983719356