FIG. 3.

Ability of Thr-to-Pro mutation at NS2A codon 149 to rescue packaging defects of two different I59 mutants. (A) Replication efficiencies of mutant RNAs in transfected BHK-21 cells. Total cellular RNA was purified 48 h postelectroporation, and accumulated viral RNAs were detected by Northern blotting using ³²P-labeled probes specific for the KUN 3′ UTR and β-actin. The efficiencies of RNA replication are expressed as percentages relative to the WT and normalized to β-actin. (B) Infectious titers of VLPs in culture fluids of tetKUNCprME-packaging BHK-21 cells electroporated with mutant repPACβgal RNAs and harvested at 3, 5, and 7 days postelectroporation. The titers were determined by infectivity assay on fresh VERO cells as described in Materials and Methods. Error bars indicate standard deviations. ^a, RT-PCR and sequencing on culture fluids revealed (pseudo)reversions to Tyr at NS2A codon 59; ^b, RT-PCR and sequencing on culture fluids revealed (pseudo)reversions to Ile NS2A codon 59; ^c, RT-PCR and sequencing on culture fluids revealed (pseudo)reversions to Trp NS2A codon 59.