The length of poly(A) and poly(U) on subgenomic mRNA 7 positive strand and negative strand, respectively.

<p>(A) Strategy for determining poly(A) tail length on coronaviral subgenomic mRNA 7 positive strand. Total cellular RNA extracted from BCoV-infected HRT cells was decapped and head-to-tail ligated. To ensure the poly(A) tail sequence is specifically derived from subgenomic mRNA 7 rather than genomic RNA, BCoV leader positive-strand-specific primer 4 (for RT) and BCoV leader (-)-strand-specific primer 3 were used for RT-PCR with ligated positive-strand coronaviral RNA as a template. The expected length of RT-PCR product is near 1.8 kb containing subgenomic mRNA 7-specific poly(A) tail. The RT-PCR product was sequenced to determine subgenomic mRNA 7 poly(A) tail length. (B) Strategy for determining poly(U) tract length on coronaviral subgenomic mRNA 7 negative strand. The method used to determine the length of poly(U) tail on negative-strand subgenomic RNA is the same as that for poly(A) tail length on positive-strand subgenomic mRNA 7 except the primer used for RT was primer 3. The expected length of RT-PCR product which contains subgenomic mRNA 7-specific poly(U) tract is ~1.8 Kb. The RT-PCR product was sequenced to determine subgenomic mRNA 7 poly(U) tract length. (C) RT-PCR product synthesized with the methods described in panels (A) and (B). The length of subgenomic mRNA 7 RT-PCR products containing poly(A) (left panel) or poly(U) (right panel) was near 1.8 kb at the different times postinfection. (D) RT-PCR control reactions. Same as described for Figure 3C, except for the use of primers 3 and 4 (not 1 and 2) in lane 1, left panel, and lane 3, right panel. (E) The length of coronaviral subgenomic RNA 7 poly(A) tail (left panel) and subgenomic RNA 7 poly(U) tract (right panel) at different time points postinfection as determined by sequencing RT-PCR products obtained in Figure 4C. M: ds DNA size markers in nt pairs. Values (E) represents the mean±SD of three individual experiments.