Abstract

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.

Highlights

  • Hepatitis delta virus (HDV)1 is the satellite virus of hepatitis B virus [1, 2], since it requires the hepatitis B virus envelope surface antigen (HBsAg) for viral particle assembly [3,4,5]

  • Deletion of the nuclear localization signal or arginine-rich motif leads to the accumulation of HDV RNA in the cytoplasm

  • The protein precipitated by anti-PKR antibodies could phosphorylate recombinant S-HDAg in vitro

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Summary

Introduction

Hepatitis delta virus (HDV)1 is the satellite virus of hepatitis B virus [1, 2], since it requires the hepatitis B virus envelope surface antigen (HBsAg) for viral particle assembly [3,4,5]. In dominant negative PKR-transfected cells, the level of phosphorylated S-HDAg was suppressed, but replication of HDV was enhanced. For Western blot analysis, about 50 ␮g of the protein was mixed with an equal volume of 2ϫ Laemmli sampling buffer, boiled for 10 min, and PKR Phosphorylates Small Delta Antigen subjected to 12% SDS-PAGE.

Results
Conclusion

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