FIG. 1.

(A) Schematic diagram of HDV cDNA constructs, RNAs, and regions amplified by the PCR. Left: thick straight bar, HDV cDNA; thin line, plasmid sequences; dashed line pHDVΔx1-NR, sequences deleted between ApaI sites (9). Direction of transcription initiated by the CMV promoter is indicated by arrows. Right: oval shapes with heavy lines, expected HDV RNA species; (+), antigenomic sense; (−), genomic sense. The location of the amber/W site is indicated by an asterisk; the genomic and antigenomic ribozyme cleavage sites (38, 41) are indicated by solid and open triangles, respectively. Small open boxes indicate locations of wild-type (pHDVx1.2-R) and mutated (pHDVΔx1-NR) polyadenylation sites. Solid bars indicate expected PCR products; arrows mark primers A (5415), B (5414), C (7646), and D (7647). Primers 7646 and 7647 correspond to sequences present only in the RNA derived from the plasmid pHDVΔx1-NR. There is a single StyI site (indicated in parentheses) in cDNAs amplified with primers 5414 and 5415 and derived from edited RNA. Primers 7646 and 7647 yield cDNAs with the same editing-sensitive site, plus an existing site that is unaffected by editing. Nonreplicating RNA is produced in cells transfected with the deletion construct pHDVΔx1-NR, which contains an ∼514-nt internal deletion and a site-directed mutation at the polyadenylation signal site. The region to be deleted is indicated by an open segment for construct pHDVx1.2-R and its derived RNAs and by a dashed line for the construct pHDVΔx1-NR. Drawings are not necessarily to scale. Horizontal gray arrows indicate transcription of RNAs from plasmid DNA templates; vertical gray arrows indicate RNA template-driven transcription occurring during HDV RNA replication. (B) Sequence of the 358-nt region analyzed by cloning and sequencing after amplification with primers 5414 and 5415. Sequence shown is antigenomic sense; numbering corresponds to the genomic RNA (39). The amber/W site is indicated by an asterisk.