FIG. 6.

HDV antigenomic RNA replication was arrested by DRB treatment and resumed after DRB withdrawal. 293T cells were transfected with both SHDAg mRNA and a dimer form of HDV antigenomic RNA at a ratio of 3:1. At 4 h posttransfection, DRB was added to cells at a concentration of 25, 50, 100, or 200 μM (lanes 5 to 8, respectively) and was refreshed every 8 h. Total RNA and protein were extracted from untreated cells at the indicated days after transfection (lanes 1 to 4) and from DRB-treated cells at 3 days posttransfection. To analyze the effect of DRB withdrawal, the drug (200 μM) was removed from transfected cells at days 0, 1, 2, and 3, and total RNA was extracted at 3 days posttransfection (lanes 9 to 12). Five micrograms of total RNA was analyzed by Northern blotting. Strand-specific HDV RNAs labeled with DIG were used as probes to detect HDV genomic RNA (gRNA), antigenomic RNA (agRNA), and mRNA. 28S/18S RNA was used as a loading control. Twenty-five micrograms of total protein was detected by Western blotting using specific antibody against RNAP II, pSer¹⁷⁷-HDAg, or HDAg.