FIGURE 4.

(A). Schematic representation of the construction of model BUNV segment AG-DBL(−2/0). This template was derived from BUN-S(ren) by the insertion of an additional NTR that had the extreme 2 nt deleted (shown by an open triangle), and resulted in the presence of two NTRs, where RNA replication could potentially initiate, designated NTRi (internal) and NTRe (external). (B) Identification of the position where replication initiated on the anti-genomic strand of AG-DBL(−2/0). Model segment AG-DBL(−2/0) 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. Labeled DNA fragments were subjected to PAGE on a sequencing gel, and bands corresponding to BUNV RNAs are marked with arrowheads. To act as size markers, RNAs generated by template AG-DBL(0/0) were analyzed alongside, and the BUN-S(ren) cDNA was sequenced using RSP. (C) The position at which RNA replication was initiated on AG-DBL(0/0) and AG-DBL(−2/0) was also analyzed in BSRT7 cells using primer extension as described above. Bands corresponding to replication products initiated at NTRi are marked with an arrowhead. (D) Metabolically labeled RNAs generated by AG-DBL(−2/0) were also subjected to agarose-urea gel electrophoresis. Replication and transcription products are marked with adjacent arrowheads. RNAs generated by parental template BUN-S(ren) are included as size markers. (E) Schematic representation of template and AG-DBL(−15/0), and identification of the position where replication initiated. The replication ability of AG-DBL(−15/0) was tested as described above. Primer extension products from templates AG-DBL(0/0), AG-DBL(−2/0), and BUN-S(ren) are included for comparison.