FIG. 1.

Rescue of recombinant BDV using different RNA polymerases for the expression of full-length antigenomic RNA from cDNA. (A) Schematic representation of the modified rescue system. Plasmids encoding the viral proteins L, N, and P under the control of the chicken β-actin promoter (CA) were transfected with either of the plasmids encoding the full-length BDV antigenome into 293T (human embryonic kidney) cells. All full-length plasmids were identical, except for the SfiI-SpeI fragment containing the promoter sequences used to control RNA expression. The promoters tested were the T7 RNA polymerase promoter (T7), the human (hPol I) or murine (mPol I) RNA polymerase I promoter, and a cytomegalovirus-derived RNA polymerase II promoter (Pol II). A BDV-specific hammerhead sequence (H) and the hepatitis delta virus ribozyme sequence (δ) were used to generate the correct 5′ and 3′ ends of the antigenomic RNA, respectively. To test the T7 RNA polymerase promoter in 293T cells, the T7 RNA polymerase (T7pol) was supplied by transfection of plasmid pCA-T7. The total amount of transfected DNA was kept constant by transfection of the empty pCA vector instead of pCA-T7 in all setups not requiring the T7 RNA polymerase. (B) Evaluation of rescue support functions of different RNA polymerases. Subconfluent 293T cells in six-well plates were transfected with 0.1 μg pCA-L, 0.5 μg pCA-N, 0.05 μg pCA-P, 1 μg pCA-T7, or pCA (empty) and 4 μg of the full-length plasmid containing the indicated promoter sequence. Three days p.t., the 293T cells were mixed with 10⁶ Vero cells and seeded onto a 94-mm dish. At 15, 22, and 29 days p.t., approximately 10⁵ cells were fixed, and rescue efficacy was evaluated by immunofluorescence analysis. The bars represent the average numbers of BDV-positive cells in three independent experiments. The standard deviations and the numbers of BDV-positive cells are indicated. (C) Northern blot analysis of cRNA synthesis from plasmids. 293T cells were transfected as described above, except that plasmid pCA-L was omitted to prevent viral RNA synthesis. To distinguish primary transcription from background signals, we transfected cells with pBRT7-HrBDVc but without pCA-T7 to prevent the synthesis of T7pol. Three days p.t., total RNA was isolated and intensely treated with Turbo DNase (Ambion) to remove transfected plasmid DNA. Ten micrograms of DNase-treated total RNA was analyzed by Northern blot using a radiolabeled DNA probe corresponding to nucleotides 4014 to 4729 of the BDV genome, representing part of the L gene. The arrow indicates the size of the BDV genome, which was determined by loading total RNA from BDV-infected cells into a slot on the same gel (not shown).