FIG 12.

Failure of precore-related proteins to form capsids or virions in human hepatoma cell culture. HepG2 cells were cotransfected with pSVB45H or pSVHBV1.5Core-, along with pCI-HBc (p21), pCI-p17, pCI-p22, pCI-p22cr, or pCI-precore (p25), as indicated. Cytoplasmic lysate and culture supernatant were harvested at day 5 posttransfection. (A) Cytoplasmic lysate (top two panels) and concentrated supernatant (bottom two panels) were resolved by SDS-PAGE, followed by immunoblotting using the HBeAg/PreC-specific MAb 1A11 or anti-precore/core (NTD) MAb T2221. (B) Cytoplasmic lysate (top two panels) and concentrated cell culture supernatant (bottom three panels) were resolved by native agarose gel electrophoresis. Following transfer to nitrocellulose membrane, HBV RNA associated with intracellular capsids was detected by a plus-strand-specific HBV riboprobe and HBV DNA in extracellular virions and naked capsids was detected by a HBV DNA probe, followed by immunoblotting using the anti-precore/core (NTD) MAb T2221 and anti-HBs polyclonal antibody (in the case of supernatant only). The different viral and subviral particles are represented schematically to the right. The wavy and straight lines inside the capsid (hexagon) indicate the pgRNA and single-stranded DNA, respectively. The small and large circles denotes the HBsAg spheres and HBV virions, respectively. p17*, artificially expressed (not processed from p25 or p22cr); S, HBs subviral particles. *, unassembled precore or core proteins.