FIG 10.
The CrPV-2 5′ UTR reduces viral fitness. (A) S2 cells were mock infected or infected with CrPV-2 or CrPV-3 (MOI of 10). Cells were metabolically labeled with [35S]methionine-cysteine for the last 30 min of infection. Lysates were subjected to SDS-PAGE, immunoblotting, or Northern blot analysis. Accumulation of viral RNA was monitored by probing for CrPV RNA genome. Expression of CrPV ORF1 was assessed by anti-RdRp antibody. (B) S2 cells were infected with CrPV-2 or CrPV-3 (MOI of 1), and viral titers were measured as described in Materials and Methods at 6, 9, 12, and 24 h p.i. **, P < 0.005; ***, P < 0.0005. Shown are averages from at least three independent experiments (± standard deviations). (C) RT-PCR analysis of the CrPV-2 and CrPV-3 5′ UTRs after serial passage in S2 cells. Cells were transfected with either CrPV-2 or CrPV-3 RNA and virus was harvested (passage 0 [P0]). Cells were then infected at an MOI of 10 and passaged in S2 cells 5 times (P1 to P5). At each passage, RNA was isolated and subjected to RT-PCR analysis.