Phosphorylation of hepatitis B virus core C-terminally truncated protein (Cp149) by PKC increases capsid assembly and stability.
The HBV (hepatitis B virus) core is a phosphoprotein whose assembly, replication, encapsidation and localization are regulated by phosphorylation. It is known that PKC (protein kinase C) regulates pgRNA (pregenomic RNA) encapsidation by phosphorylation of the C-terminus of core, which is a component packaged into capsid. Neither the N-terminal residue phosphorylated by PKC nor the role of the C-terminal phosphorylation have been cleary defined. In the present study we found that HBV Cp149 (core protein C-terminally truncated at amino acid 149) expressed in Escherichia coli was phosphorylated by PKC at Ser(106). PKC-mediated phosphorylation increased core affinity, as well as assembly and capsid stability. In vitro phosphorylation with core mutants (S26A, T70A, S106A and T114A) revealed that the Ser(106) mutation inhibited phosphorylation of core by PKC. CD analysis also revealed that PKC-mediated phosphorylation stabilized the secondary structure of capsid. When either pCMV/FLAG-Cp149[WT (wild-type)] or pCMV/FLAG-S106A Cp149 was transfected into Huh7 human hepatoma cells, mutant capsid level was decreased by 2.06-fold with the S106A mutant when compared with WT, although the same level of total protein was expressed in both cases. In addition, when pUC1.2x and pUC1.2x/S106A were transfected, mutant virus titre was decreased 2.31-fold compared with WT virus titre. In conclusion, PKC-mediated phosphorylation increased capsid assembly, stability and structural stability.
Kang
Hang
H
School of Biological Sciences, Seoul National University, Shillim-dong, Seoul 151-742, Korea.
Yu
Jaehoon
J
Jung
Guhung
G
D002213:Capsid / Q000502:physiology*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002942:Circular Dichroism; D006512:Hepatitis B Core Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006515:Hepatitis B virus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D017433:Protein Structure, Secondary; D020349:Surface Plasmon Resonance; D014407:Tumor Cells, Cultured
D006512:Hepatitis B Core Antigens; D011493:Protein Kinase C