FIG.3.

Template switching from an alternate internal DI RNA donor sequence to 5′-proximal genomic acceptor sites and involvement of viral sgmRNA 7 in formation of novel sgmRNAs. (A) Structure of Wt^N DI RNA and mutant derivatives Wt^N-M1 and Wt^N-M2. In Wt^N, the 197-nt insert contains the 190-nt sgmRNA 7 IS region of BCoV (genomic nt 29,324 to 29,513). (B) RdRp template switch site and Northern analysis results for Wt^N DI RNA containing the intergenic sequence region for sgmRNA 7. Note that the sgmRNA contained a full-length leader. (C) RdRp template switch sites for Wt^N-M1 DI RNA that yielded sgmRNAs of two types. (a) The first type resulted from an acceptor site within the DI RNA or viral genomic leader and yielded an sgmRNA with a 34-nt minileader. The structure of the sgmRNA is shown. (b) The second type resulted from a template switch in trans involving the 5′-proximal region of helper virus sgmRNA 7. The structure of the novel sgmRNA is shown, with the unique sgmRNA 7-specific nucleotides identified with triangles. (D) RdRp template switch sites for Wt^N-M2 DI RNA that yielded sgmRNAs of two types. (a) The first type resulted from an acceptor site within the DI RNA or viral genomic leader and yielded an sgmRNA with a 60-nt minileader. The structure of the sgmRNA is shown. (b) The second type resulted from a template switch in trans involving the 5′-proximal region of helper virus sgmRNA 7. The structure of the novel sgmRNA is shown, with the unique sgmRNA 7-specific nucleotides identified with triangles. (E) Electrophoresis of RT-PCR products in a gel of 3.0% agarose.