FIG. 5.

The carboxyl terminus of HBcAg is exposed on the surface of DP rcDNA-containing nucleocapsid. (A) A rabbit polyclonal antibody (HBc170) was raised against the synthetic peptide corresponding to the carboxy-terminal 14 amino acid residues of the HBV core protein. The underlined 12-amino-acid peptide within the C-terminal 14 amino acid residues is one of the bipartite NLSs of HBcAg. (B) Proteins in cell lysates of HepDES19 cells cultured in the presence or absence of tetracycline for 8 days were resolved by SDS-polyacrylamide gel electrophoresis, transferred onto the membrane, probed with the antibody HBc170, and visualized by Li-COR. A cross-reactive cellular protein band is indicated with an asterisk, and β-actin served as the loading controls. (C) Cytoplasmic lysate of HepDES19 cells were subjected to immunoprecipitation with antibodies against the HBV core protein (Dako), C-terminal 14-amino-acid peptide (HBc170), and HBsAg. Core DNA and DP DNA were extracted with or without prior DNase I digestion from the original lysate (input) and immunocomplexes on beads (IP) and analyzed by Southern blot hybridization. Lanes 1 and 2 were loaded with 1/10 of core DNA from the cytoplasmic fraction of one 60-mm dish; lanes 3 to 14 were loaded with half of the indicated DNA samples from the cytoplasmic fraction of one 60-mm dish. The positions of rcDNA (RC) and single-stranded DNA (SS) are indicated. MW, molecular size.