FIGURE 1.

(A) Schematic representation of the BUNV S segment and its RNA replication ability. The annealing position of oligonucleotide RSP used to detect the 5′ end of the genome is shown. The anti-genomic 5′ NTR encompasses S segment nucleotides 1–85 and is shaded. The anti-genomic 3′ NTR encompasses S segment nucleotides 788–961 and is shown as an open box. (B) Schematic representation of model BUNV segment AG-DBL(0/0). This template was derived from BUN-S(ren) by the insertion of an additional anti-genomic 3′ NTR, resulting in two identical 3′ NTRs where replication could potentially initiate, designated NTRi (internal) and NTRe (external). (C) Identification of the position where replication initiated on the anti-genomic strands of AG-DBL(0/0). Model segment AG-DBL(0/0) was expressed in BHK-21 cells along with BUNV S and L ORFs (+), or the S ORF alone (−). The resulting BUNV RdRp-specific RNAs were analyzed by primer extension using end-labeled oligonucleotide RSP (lanes 1–4). Labeled DNA fragments were subjected to denaturing PAGE on a sequencing gel. Bands corresponding to either corrected or uncorrected negative-sense replication products are indicated with arrowheads. To act as size markers, RNAs generated by parental template BUN-S(ren) were analyzed alongside, and the corresponding cDNA was sequenced using oligonucleotide RSP. To eliminate a role of vaccinia virus-mediated effects in the repair process, the template AG-DBL(0/0) was also generated in BSRT7 cells, and its replication ability analyzed by primer extension as described above (lanes 5,6). (D) Metabolically labeled RNAs generated by AG-DBL(0/0) were also subjected to agarose-urea gel electrophoresis. Replication and transcription products are marked with adjacent arrowheads, and uncorrected RNA products are marked with an asterisk. RNAs generated by parental template BUN-S(ren) are included as size markers.