FIG. 1.

Scheme for measuring high-frequency template switching. (A) Structure of Wt^12.7 DI RNA, generated from MluI-linearized pWt^12.7 by T7 RNA polymerase, and its mutant derivative, Wt^X. The wt BCoV DI RNA is composed of the two ends of the viral genome joined at nt 494 and 29,392 and, for the formation of pWt^12.7, has placed within it an in-frame 30-nt reporter sequence from the porcine transmissible gastroenteritis coronavirus N gene, a 199-nt insert containing the 191-nt BCoV intergenic sequence (IS) region from upstream of the open reading frame for sgmRNA 5 (genomic nt 27,967 to 28,157), and a 94-nt insert containing a 92-nt reporter sequence from the HSV gD gene (29). Template switch donor sites X, IS1, IS2, and IS3 are underlined. In Wt^X DI RNA, sites IS1, IS2, and IS3 have been made nonfunctional as donor sites. The 22-nt donor region within which all other mutations of pWt^X were made for this study is shown. (B) Overall experimental scheme. (C) Structure of the 33-nt minileader on sgmRNA compared to the wt 65-nt BCoV leader.