FIG.6.

Accumulation of processed DNA-directed antigenomic HDV RNA transcripts. Panel A indicates the region of an expression vector containing greater-than-unit-length sequences of HDV. Following transfection into 293TRex cells, these sequences should be transcribed by Pol II as an antigenomic HDV RNA transcript. The 5′ end corresponds to the known 5′ end of the HDV mRNA. By use of the known poly(A) signal (small open box), this RNA can be processed to release an mRNA comparable to the natural HDV mRNA. Downstream of the poly(A) signal is a unit-length antigenomic RNA sequence flanked by two copies of the antigenomic ribozyme (small open circle). This region of the RNA can be ribozyme processed to release unit-length linear antigenomic RNA, some of which can be further processed by ligation to become unit-length antigenomic RNA circles, like those that are detected during natural HDV replication. It should be noted that the unit-length sequence within the primary transcript does not contain an active poly(A) signal (having been inactivated by conversion of AAUAAA to UUUAAA [34]). Also, the δAg ORF within this sequence is inactivated (by a 2-nucleotide deletion). Three versions of the expression vector were used, and results are shown in panels B to D. The differences were all in terms of the δAg ORF at the 5′ end of the antigenomic RNA transcript. In panel B, the ORF was inactivated by a 2-nucleotide deletion. In panel C, the ORF was subjected to a 36-nucleotide in-frame deletion that produces a protein that cannot support HDV genome replication but is able to support the processing and accumulation of DNA-directed HDV RNA species (25, 32). In panel D, the ORF is the wild-type small δAg, which supported HDV RNA-directed RNA replication. Cells were transfected with each of these specific expression constructs. At 4 h, the cultures were reseeded as identical smaller cultures, and measurements were taken at daily time points up to 6 days, as indicated. For each culture, total RNA was extracted and examined by Northern assay to quantitate both the mRNA (filled circles) and the unit-length antigenomic RNA (open circles). Panel E represents a variation of the experiment described for panel C. At 24 h after the transfection, the cells were reseeded in the indicated concentrations of amanitin. After another 24 h, total RNA was extracted and assayed for the accumulation of the two processed HDV RNA species. The amounts of mRNA and unit-length antigenomic RNAs are indicated by filled and open bars, respectively. These data are normalized relative to those cells not exposed to amanitin.