Figure 4

Transcription profiles of HBV pgRNA during cell growth to confluence. (A) Schematic representation of the strategy for detection of origins of viral transcripts. 1.3ES2 cells contain a 1.3-fold HBV genome with the Bcl I genetic marker in the 3' terminus redundancy. Viral transcripts from the integrated genome generate a Bcl I marker within the 3' terminus redundancy region, while transcripts derived from cccDNA does not. After RT-PCR (with primers HBV2338/F and T20-Taq/HBV5) and restriction enzyme digestion with Ssp I and Bcl I, the restricted PCR products are separated by agarose gel electrophoresis. The existence of 332-bp product, which lacks the Bcl I site, represents transcripts from cccDNA, whereas the two fragments of 200-bp and 132-bp represent transcripts from the integrated genome. (B) The cccDNA was collected with the Hirt extraction method at each time point, thermally denatured at 85°C for 5 min, and then subjected to Eco RI digestion before electrophoresis on a 1.2% agarose gel. All the lanes were loaded with samples representing the same numbers of cells. The autoradiograph shows the cccDNA and ssDNA; the line chart shows the relative quantitative intensities of the cccDNA. (C) Equal amounts of total RNA from each sample were used for RT-PCR with the primer pair HBV2338/F and T20-Taq/HBV5. Following Ssp I/Bcl I double digestion, the restricted PCR products were separated by electrophoresis. The relative quantitative intensities of the PCR products are shown in the line chart: 332-bp (--●--), 200-bp and 132-bp (...○...), and 603-bp (--▼--). (D) Equal amounts of the HBV RT-PCR products, amplified by the primer pair HBV2338/F and T20-Taq/HBV5, were subjected to Ssp I/Bcl I double digestion, and the restricted PCR products were separated by electrophoresis. The relative quantitative intensities of the PCR products are shown in the line chart: 332-bp (--●--), 200-bp and 132-bp (...○...).