Secretion of Genome-Free Hepatitis B Virus – Single Strand Blocking Model for Virion Morphogenesis of Para-retrovirus
Figure 4
Quantitative analyses of DNA-filled and empty virions secreted by WT HBV in vitro and in vivo.
HBV virions in the sera of experimentally infected chimpanzees (lanes 1–7; 2 µl each serum) and purified from WT HBV transfected HepG-2 cells by CsCl density gradient centrifugation (lanes 15–18; 30 µl each fraction), along with known amounts of HBV capsid standard (1–32 ng; lanes 8–13), were resolved by SDS-PAGE; Virion-associated core protein was detected by western blot analysis using the anti-HBV core monoclonal antibody (top image). The virion DNA released by SDS-protease digestion from the same chimpanzee sera (lanes 1–7; 2 µl each serum) and HepG-2 virion fractions (lanes 15–18; 2 µl each fraction) were also resolved on an agarose gel, along with known amounts of HBV DNA (3 kb, 2.5–160 pg, lanes 8–14), and detected by Southern blot analysis (bottom image). Lane 1 was from chimpanzee A0A006 at week 7 post-infection (PI); lanes 2 & 3, chimpanzee 1603 at week 15 and 16 PI, respectively; lanes 4 & 5, chimpanzee 1618 at week 14 and 17; lanes 6 & 7, chimpanzee 1616 at week 20 and 23 PI. The amounts of HBV core protein (top) and virion DNA (bottom) are indicated at the bottom of the lanes. The percentages of DNA-containing virions, calculated as the molar amounts of HBV DNA divided by the molar amounts of HBV capsid (240 copies of core subunits per capsid) and multiplied by 100, are also indicated. Please note that 15-fold more HepG-2 virions were loaded on the SDS-PAGE gel than on the agarose gel, which was taken into account in the calculation of DNA-containing virions. F#, virion fraction number from the CsCl gradient. C, core protein; RC and DSL, RC and DSL DNA; X, blank lane (lane 14) on the SDS-PAGE gel.