FIG. 2.

Immature and mature secretions of Dane particles in HBV variants and wt of different subtypes in gradient centrifugation analysis. Ten micrograms of subtype adr or ayw plasmid DNA was transfected into human hepatoma HepG2 or Huh7cells, respectively. The media were collected on days 5 and 7 posttransfection. Viral particles were then purified from the media through a 20% sucrose cushion and subjected to isopycnic centrifugation in a gradient of 20 to 50% (wt/vol) cesium chloride. Fractions were separated according to their buoyant density and then submitted to HBsAg assay using an AbbottAuszyme EIA kit and HBeAg assay using an Abbott HBe (rDNA) EIA kit (data not shown). The enveloped virions, which are HBsAg positive, band at a density near 1.24 g/cm³ around fractions 10 to 14, while the nonenveloped core particles, which are HBsAg negative and HBcAg positive, band at a density near 1.35 g/cm³ around fractions 2 to 6. The focus of this study is on enveloped virion fractions. Extracellular HBV DNA in every other fraction was detected by a 3.1-kb HBV ayw subtype (upper panel) or adr subtype (lower panel) DNA probe. To reveal the weak HBV DNA signals at fractions 10 and 12 of ayw mutant F97I (upper right panel), prolonged exposure of the X-ray film was necessary, which resulted in the background noise at fractions 16, 18, and 20. RC, full-length relaxed-circle HBV DNA replicative intermediates at 4.0 kb; SS, full-length single-stranded HBV DNA replicative intermediates at 1.5 kb. Based on four independent transfection experiments, the RC/SS ratio of adr mutant I97L in HepG2 cells is close to 1.5 ± 0.4, while the RC/SS ratio of adr wt I97I in HepG2 cells is close to 9.3 ± 2.4. These RC/SS ratios are approximately twofold higher than the previously reported RC/SS ratios of ayw wt F97F and ayw mutant F97L in Huh7 cells (see Discussion).