Kinetics of DHBV RNA transcription, DNA replication and cccDNA formation in DSL212 cells.

<p>DSL212 cells were seeded in 6-well plates and cultured in the presence of tetracycline (1 μg/ml) until cell monolayers became confluent. Cells were then cultured in media without tetracycline and harvested at the indicated days since the removal of tetracycline. Total cellular RNA, cytoplasmic core DNA and total cellular Hirt DNA were extracted and analyzed by Northern and Southern blot hybridization, respectively. (A) For viral RNA analysis, each lane was loaded with 5 μg of total RNA. pgRNA, pregenomic RNA; sRNA, mRNAs specifying the two envelope proteins. Ribosomal RNA (28S and 18S) served as loading controls. For DHBV core DNA (B) and Hirt DNA (C) analysis, each lane represents the amount of viral DNA extracted from one half of cells in a well of 6-well plate. RC, relaxed circular DNA; DSL, double stranded linear DNA; SS. single stranded DNA; cccDNA, covalently-closed circular DNA. Unit length of linear DHBV DNA (lane 10) and core or Hirt DNA extracted from dstet5 cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043270#pone.0043270-Guo4" target="_blank">[40]</a> cultured in the absence of tetracycline for 8 days (lane 11) served as controls.