The double-stranded RNA-activated kinase, PKR, can phosphorylate hepatitis D virus small delta antigen at functional serine and threonine residues.
Hepatitis D virus (HDV) encodes two proteins, the 24-kDa small delta antigen (S-HDAg) and 27-kDa large delta antigen (L-HDAg) in its single open reading frame. Both of them had been identified as nuclear phosphoproteins. Moreover, the phosphorylated form of S-HDAg was shown to be important for HDV replication. However, the kinase responsible for S-HDAg phosphorylation remains unknown. Therefore, we employed an in-gel kinase assay to search candidate kinases and indeed identified a kinase with a molecular mass of about 68 kDa. Much evidence demonstrated this kinase to be the double-stranded RNA-activated kinase, PKR. The immunoprecipitated endogenous PKR was sufficient to catalyze S-HDAg phosphorylation, and the kinase activity disappeared in the PKR-depleted cell lysate. The S-HDAg and PKR could be co-immunoprecipitated together, and both of them co-located in the nucleolus. The LC/MS/MS analysis revealed that the serine 177, serine 180, and threonine 182 of S-HDAg were phosphorylated by PKR in vitro. This result was consistent with previous phosphoamino acid analysis indicating that serine and threonine were phosphorylation targets in S-HDAg. Furthermore, serine 177 was also shown to be the predominant phosphorylation site for S-HDAg purified the from cell line. In dominant negative PKR-transfected cells, the level of phosphorylated S-HDAg was suppressed, but replication of HDV was enhanced. Other than human immunodeficiency virus type 1 trans-activating protein (Tat), S-HDAg is another viral protein phosphorylated by PKR that may regulates HDV replication and viral response to interferon therapy.
Chen
Chi-Wu
CW
Graduate Institute of Microbiology and Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.
Tsay
Yeou-Guang
YG
Wu
Hui-Lin
HL
Lee
Chi-Hua
CH
Chen
Ding-Shinn
DS
Chen
Pei-Jer
PJ
D002460:Cell Line; D002466:Cell Nucleolus / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D005799:Genes, Dominant; D005822:Genetic Vectors; D006367:HeLa Cells; D018963:Hepatitis Antigens / Q000737:chemistry* / Q000378:metabolism*; D035923:Hepatitis delta Antigens; D006801:Humans; D016898:Interferon-alpha / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D012694:Serine / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*; D014162:Transfection; D019892:eIF-2 Kinase / Q000378:metabolism*
D018076:DNA, Complementary; D018963:Hepatitis Antigens; D035923:Hepatitis delta Antigens; D016898:Interferon-alpha; D010455:Peptides; C442244:hepatitis delta virus large antigen; D013912:Threonine; D012694:Serine; D019892:eIF-2 Kinase