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. 2005 Jan;79(2):732–744. doi: 10.1128/JVI.79.2.732-744.2005

FIG. 4.

FIG. 4.

Temperature sensitivity of viral RNA synthesis in ts53 virus-infected cells. Primer extension assays were performed with 4 μg of total RNAs and vRNA- or mRNA/cRNA-specific DNA primers labeled at their 5′ end with [γ-32P]ATP. Reverse-transcribed products were analyzed on a 5% polyacrylamide gels containing 7 M urea in TBE buffer and were detected by autoradiography. (A) Dose response in the primer extension assay. Total RNAs were purified from WSN/33 virus-infected MDCK cells at 6 h postinfection (hpi). Primer extension assays were performed with 1 (lane 1), 2 (lane 2), 4 (lane 3), and 8 μg (lane 4) of total RNA and a primer specific for segment (Seg.) 3 mRNA/cRNA as described in Materials and Methods. To show that the amount of the primer was in excess in the primer extension assay, 1/400 (lane 5), 1/200 (lane 6), and 1/100 (lane 7) of the input primers were also subjected to electrophoresis. (B) Synthesis of viral RNAs in ts53 virus-infected cells. MDCK cells were infectedwith WSN/33 or ts53 virus at an MOI of 10. At 3 (lanes 2, 6, 10, and 14), 6 (lanes 3, 7, 11, and 15), and 9 (lanes 4, 8, 12, and 16) h postinfection at 34°C (lanes 1 to 4 and 9 to 12) or 39.5°C (lanes 5 to 8 and 13 to 16), cells were collected and total RNAs were isolated. Primer extension assays were performed with primers specific for segment 3 vRNA or mRNA/cRNA. Total RNAs prepared from uninfected cells were also analyzed (lanes 1, 5, 9, and 13). As an internal control, luciferase mRNA (10 ng) was added to each sample, and the sample was then subjected to primer extension assays. (C) Viral mRNA synthesis in the presence of a protein synthesis inhibitor. MDCK cells were infected with WSN/33 or ts53 virus at an MOI of 10 in the presence or absence of 100 μg of cycloheximide (CHX)/ml. At 6 h postinfection in the presence (lanes 3, 6, 9, 12, 15, 18, 21, and 24) or absence (lanes 2, 5, 8, 11, 14, 17, 20, and 23) of CHX at 34 or 39.5°C, total RNAs were isolated. Primer extension assays were performed with primers specific for vRNA and mRNA/cRNA of segment 3 and segment 7. Total RNAs prepared from uninfected cells without CHX were also analyzed (lanes 1, 4, 7, 10, 13, 16, 19, and 22). luciferase mRNA was used as an internal control for the whole procedure.