FIG. 4.

Enzymatic assay for enveloped HBV virion formation in PTHs. (A) Dose dependence. Particles in supernatants from PTHs transduced with the indicated MOIs of Ad-HBV1.3 and collected on days 9 and 10 p.i. were PEG precipitated. Naked DNA and DNA in nonenveloped particles was digested by pronase plus DNase. DNA from enveloped particles was subsequently released by proteinase K-SDS treatment. Aliquots were loaded directly (lanes −) or after digestion with EcoRI (E. RI) (lanes +) and detected with a DIG-labeled HBV probe. M, DIG-labeled DNA marker with fragment sizes in kilobases; St, linear 3.2-kb HBV genome. The positions of HBV RC-DNA and linear (Lin) DNA and of the assignable HBV and Ad-HBV1.3 EcoRI fragments are indicated on the right. The arrows denote bands in the undigested samples whose exact natures are not known. (B) Time course. Supernatants from PTHs transduced with Ad-HBV1.3 at an MOI of 100 were collected every 2 days as indicated, except for the day 18 sample, which was collected over 4 days. Extracellular DNA was analyzed as for panel A. An aliquot from the day 6 sample was digested with XhoI (lane d6X Xho), which linearizes the circular HBV genome and generates a 1.7- plus a 1.5-kb fragment from linear molecules. Ad-HBV1.3 produces two HBV-containing fragments of 4.3 and 2.7 kb. St, 50 pg of linear 3.2-kb HBV genome.