FIG. 7.

RNA synthesis in CS1 mutants. (A) Metabolic labeling of viral RNA. DBT cells were mock infected (M) or infected with A59, icwt, or the indicated CS1 mutants. At 4.5 hpi, actinomycin D was added to a final concentration of 20 μg/ml. Cells were radiolabeled with [³H]uridine from 5 to 7 hpi and lysed, and TCA-precipitated viral RNA was quantitated by liquid scintillation counting. Error bars represent standard deviations of duplicate measurements. mutΔ indicates mutΔCS1 throughout the figure. (B) Northern blot analysis. DBT cells were mock infected or infected with A59, icwt, or the indicated CS1 mutants in parallel with the infections shown in panel A. Cells were lysed in Trizol at 10 hpi, and RNA from approximately 4 × 10⁵ cells was separated on a 0.8% agarose-formaldehyde gel. RNA was transferred to a nylon membrane, UV cross-linked, and probed with a ³²P-labeled negative-polarity primer complementary to the 3′ UTR to detect positive-strand RNA species. Top, 6-h exposure of genomic RNA (RNA 1); bottom, 1-h exposure of all positive-strand RNA species. Genomic RNA and sgRNA species are indicated by number to the right.