FIG. 2.

Replication abilities and ratios of positive-strand RNA to negative-strand RNA of BVDV DI9c ORF mutants. (A) DI9c ORF derivatives (the same set of mutants analyzed in Fig. 3, 5, 6, and 7) were transfected into BHK-21 or MDBK cells. Identical numbers of cells were harvested at the indicated time p.t., and the cytoplasmic fractions were analyzed for newly synthesized positive-strand viral RNA by a quantitative RPA with ³²P-labeled antisense probe A (see Materials and Methods and Fig. 4A). The protected fragments were separated on a 5% polyacrylamide gel containing 7 M urea and quantitated by phosphorimaging. (Top) RPA carried out with cytoplasmic RNA of transfected BHK-21 cells at 24 h p.t. Lanes: as, aliquot of input antisense probe; pc, RPA performed on 300 ng of in vitro-transcribed DI9c cRNA (positive control); wt, RPA with cytoplasmic RNA from wild-type DI9c transfected cells; italic numbers, identical experiments carried out with DI9c ORF mutants 7, 12, 13, 15, and 16 to 21 (numbering scheme as in Fig. 1 and Table 2). Mutant 15 was applied as a negative control. The protein-coding regions of DI9c which contain the various mutations are indicated. By quantitative evaluation of five independent transcription-transfection experiments, the relative replication abilities of mutants 12 and 13 were determined to correspond 63% ± 12% and 44% ± 14% of that of the wild-type replicon, respectively. (Middle and bottom) RPA of DI9c derivatives 7 and 15 to 21 at 48 h after transfection into BHK-21 and MDBK cells. Mutant 7 (replicating at 10% ± 4% of the efficiency of the wild-type DI9c) was used as a reference in these experiments because this RNA does not induce a CPE at this time point (for a quantitative analysis, see Fig. 5 and 6). Note that except for two RNAs (1b and 1c), which were analyzed in detail in reference 12, the transfection experiments with all DI9c ORF derivatives listed in Fig. 1 and Table 2 yielded negative results, i.e., blank gels at 24 h as well as at later time points p.t. (these data are summarized in Table 3). The latter mutants were also negative if tested by RT-PCR (data not shown). (B) Using sense and antisense probes during RPA, the ratio of progeny positive-strand RNA to negative-strand RNA intermediate was measured with all replication-competent DI9c ORF derivatives (1b, 1c, 7, 12, 13, and 17) and compared with the wild-type (wt) value. Error bars indicate the mean deviations of five independent transcription-transfection experiments.