FIG. 2.

Replication and packaging of NS2A 59 pseudorevertants. (A) Hydrophobicity plot of amino acid residues found in pseudorevertants and cloned into NS2A codon 59 of repPACβgal cDNA. (B) Northern blot analysis of total cellular RNA from BHK-21 cells 2 days postelectroporation with various mutant KUN replicon RNAs. The probes were ³²P-labeled cDNA fragments specific for the KUN 3′ UTR and for β-actin. Efficiencies of replication are expressed as percentages of accumulated viral RNAs relative to WT and normalized to β-actin (shown under the blots). (C) 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 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 Ile and Tyr at NS2A codon 59; ^b, RT-PCR and sequencing on culture fluids revealed (pseudo)reversions to Ile and Val at NS2A codon 59.