FIG. 5.

Replication of RNAs and synthesis of viral proteins in cells transfected with PaV cDNA clones. (A) BHK-21 cells were infected with vTF7-3 at an MOI of 10 PFU/cell. One hour postinfection, the cells were transfected with 5 μg of pPaV1(0,0) (lane 1), 2.5 μg each of pPaV1(0,0) and pPaV2(0,0) (lane 2) or 1 μg of PaV virion RNA (lane 3) and incubated at 28°C. After 22 h of incubation, actinomycin D was added at 5 μg/ml; 30 min later, replicating RNAs were metabolically labeled by incorporation of [³H]uridine (20 μCi/ml) for 4 h before total cellular RNA was harvested. RNAs were resolved by electrophoresis on a 1% agarose-formaldehyde gel and visualized by fluorography. PaV RNA1, RNA2, and RNA3 are identified on the left. (B) BHK-21 cells were infected with vTF7-3 at an MOI of 10 PFU/cell. One hour postinfection, the cells were transfected with water (lane 1), 1 μg of PaV virion RNA (lane 2), or 2.5 μg each of pPaV1(0,0) and pPaV2(0,0) (lane 3) and incubated at 28°C. After 44 h of incubation, actinomycin D was added at 5 μg/ml and incubation continued. Following a 0.5-h preincubation in methionine-cysteine-free medium, 48 h posttransfection proteins were labeled with [³⁵S]methionine-cysteine for a period of 2 h. Cytoplasmic extracts were harvested and resolved by SDS-PAGE on a 12.5% gel, and the labeled proteins were visualized by autoradiography. PaV proteins A and α are identified on the right.