FIG. 3.

Polyadenylation of B19V RNAs shows a shift from (pA)p early in infection to (pA)d late during infection of CD36⁺ EPCs or UT7/Epo-S1 cells. In addition, B19V RNAs generated following transfection of nonreplicating B19V genomes did not generate significant levels of B19V RNAs polyadenylated at (pA)d. CD36⁺ EPCs were infected with B19V, and total RNAs were isolated at 18, 24, 36, and 72 h p.i.; UT7/Epo-S1 cells were infected with B19V, and total RNAs were isolated at 18, 36, and 72 h p.i. Plasmids pM20, pM20NS(−), and pSKB19NSCap were transfected into UT7/Epo-S1 cells, and 72 h later, total RNA was isolated. All RNA samples were protected by probe 8, probe 6, or probe 10, and results for a representative experiment are shown. (A) RPA using probe 8. Bands representing RNAs that either read through or were polyadenylated at (pA)p1, (pA)p2, and (pA)d are designated RT(pA)p1, RT(pA)p2, and RT(pA)d or (pA)p1, (pA)p2, and (pA)d, respectively. Quantification of the ratio of B19V RNA at (pA)d to total polyadenylated B19V RNA is shown as a percentage [(pA)d%]. (B) RPA using probe 6. Bands representing RNAs that were either spliced or unspliced at the A2-1 acceptor are designated Unspl (for unspliced RNAs) and Spl (for spliced RNAs). Alternative representations for these bands are shown with their coding capabilities in parentheses. Quantification of the ratio of spliced RNA (VP2 mRNA) versus unspliced RNA (VP1 mRNA) is shown as the Spl/Unspl ratio, and quantification of the VP2 mRNA is shown as a percentage of VP2 mRNA (VP2 mRNA%) in the total RNA protected by probe 6. The identities of the bands indicated with a star are unknown, as previously described (46), and therefore they were not included in the quantification. (C) RPA using probe 10. Bands representing RNAs that were either spliced or unspliced at A2-2 are designated Unspl (for unspliced RNAs) and Spl (for spliced RNAs). Alternative representations for these bands are shown with their coding capabilities in parentheses. Quantification of the ratio of spliced RNA (11-kDa protein mRNA) versus unspliced RNA (VP2 mRNA) is shown as the Spl/Unspl ratio. The bands indicated with a star are likely nonspecific hybridized products. (D) B19V RNAs were stable. CD36⁺ EPCs were infected with B19V. After 72 h, cells were treated with actinomycin D, and total RNAs were isolated at the times indicated and were protected by probe 8. All of the quantifications represent average results for at least three individual experiments, with standard deviations underneath. N, not determined because there were nearly no spliced RNAs detected. Lanes 1 in panels A, B, and C, a ³²P-labeled RNA ladder with the respective sizes indicated to the left, as previously described (32).