FIG. 3.

Analysis of positive- and negative-strand viral RNA accumulation and positive-strand RNA stability in wild-type- or Δ3′ NCR-infected cells. For the RNase protection analysis shown in panels A and B, RNA samples were derived from the experiment displayed in Fig. 2. (A) Positive-strand viral RNA accumulation was determined in a standard RNase protection assay as described in Materials and Methods. Marker RNAs (with sizes indicated in nucleotides on the left side of the panel) are shown in lane 1. A 5.3-fmol aliquot of undigested probe was used for comparison to quantitate the molar amounts of RNA loaded in each lane by phosphorimager analysis (lane 2). Equal amounts (108 ng) of total cellular RNA were protected by 25 fmol of probe complementary to positive-sense poliovirus RNA sequences 5601 to 5809 (lanes 3 to 16). HeLa cells (lane 3 to lane 9) or SK-N-SH cells (lane 10 to lane 16) were infected with either wild-type poliovirus (lanes 3 to 5 and 10 to 12) or Δ3′ NCR PV1 (lanes 6 to 8 and 13 to 15). The RNA harvest times (hours postinfection) are indicated at the top of each lane. Mock-infected cells were harvested at 8 h postinfection (lanes 9 and 16). (B) Negative-strand RNA levels were determined by subjecting 1.3 μg of total cellular RNA to a two-cycle RNase protection assay as described in Materials and Methods. RNA samples were separated on 7 M urea-8% polyacrylamide gels. (C) RNA stability of positive-strand RNAs derived from infection of monolayers of SK-N-SH cells with wild-type or Δ3′ NCR mutant virus. Cells were infected at an MOI of ∼20 in the presence of 2 mM guanidine-HCl, and total RNA was isolated at the times indicated as described in Materials and Methods. Glyoxal-treated RNA (10 μg) was subjected to Northern blot analysis following electrophoresis on a 1.0% agarose gel in the presence of 10 mM sodium phosphate. Poliovirus and β-actin RNAswere detected by hybridization to ³²P-labeled random hexamer-primed DNA probes. Hybridization and washing conditions were as described elsewhere (9).