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. 2005 Mar;79(6):3586–3594. doi: 10.1128/JVI.79.6.3586-3594.2005

FIG. 2.

FIG. 2.

Schematic representation of RNA templates BUN-S(ren) and AG-CB and analysis of their RNA synthesis activities. (A) The first 25 nt of both 3′ and 5′ NTRs of BUN-S(ren) are sufficient to signal transcription. RNAs were generated in vTF7-3-infected BHK cells transfected with the corresponding genome analog expressing cDNA and either BUNV S and L support plasmids (pL) (+) or S support plasmid alone (−). Bands representing mRNAs (vertical bar), T7 RNA polymerase primary transcripts, and BUNV-specific antigenomic RNAs (arrowheads) are marked. Template BUN-S(ren)-25/25 is identical to template BUN-S(ren), except that it contains only the terminal 25 nt of 3′ and 5′ NTRs. (B) Representation of the first 25 nt of both 3′ and 5′ NTRs of the genomic and antigenomic strands ofBUN-S(ren) and the genomic strand of AG-CB. The AG-CB antigenome is not shown, as it is identical in sequence to the AG-CB genomic strand for the nucleotides shown. Nucleotide differences from the genomic strand of BUN-S(ren) are shaded, and the potential to form Watson-Crick base pairs (*) or noncanonical U-G pairings (•) are also shown. Template AG-CB was derived from BUN-S(ren) by making nine nucleotide changes to the corresponding cDNA. (C) The RNA synthesis characteristics of BUN-S(ren) and AG-CB were compared by direct visualization of metabolically labeled actinomycin D-resistant RNAs using agarose-urea gel electrophoresis. This gel system allows separation of the three BUNV-specific RNAs, due to its ability to resolve RNAs based on both size and nucleotide composition. (D) RNAs generated by the genomic strands of BUN-S(ren) and AG-CB were also detected using primer extension analysis with the negative-sense oligonucleotide BUNMRNA. These RNAs were generated in vTF7-3-infected BHK cells transfected with the corresponding genome analog expressing cDNA and either BUNV S and L support plasmids (+) or S support plasmid alone (−). Bands representing mRNAs (vertical bar), T7 RNA polymerase primary transcripts, and BUNV-specific antigenomic RNAs (arrowheads) are marked.