TABLE 3.
Functional analysis of BVDV DI9c ORF mutantsa
| Mutant | Replicationb | Processingc | Complementabilityd |
|---|---|---|---|
| NS3 | |||
| 1a | − | a | − |
| 1b | + | wt | − |
| 1c | + | wt | − |
| 1d | − | a | − |
| 2 | − | a | − |
| 3 | − | a | − |
| 4 | − | a | − |
| 5 | − | a | − |
| 6 | − | a | − |
| 7 | + | wt | − |
| 8 | − | a | − |
| 9 | − | a | − |
| 10a | − | a | − |
| 10b | − | a | − |
| 10c | − | a | − |
| NS4A | |||
| 11 | − | a | − |
| NS4B | |||
| 12 | + | wt | − |
| 13 | + | wt | − |
| 14 | − | wt | − |
| 15 | − | a | − |
| NS5A | |||
| 16 | − | wt | + |
| 17 | + | wt | + |
| 18 | − | wt | + |
| 19 | ± | wt | + |
| 20 | − | a | − |
| 21 | − | a | − |
| NS5B | |||
| 22 | − | a | − |
| 23 | − | wt | − |
| 24 | − | wt | − |
| 25 | − | wt | − |
| 26 | − | wt | − |
| 27 | − | wt | − |
| Cleavage sites | |||
| 28 | − | a | − |
| 29 | − | a | − |
| 30 | − | a | − |
| 31 | − | a | − |
All RNAs were generated by in vitro transcription and tested as described in the text. Importantly, the diverse DI9c ORF derivatives were confirmed by RPA and RT-PCR to exhibit the same stability as the wild-type replicon under the experimental conditions of the replication and translation assays (data not shown). RT-PCR on cytoplasmic RNA preparations of transfected cells followed by sequencing or restriction analysis of the resulting PCR products was performed to exclude reversion or contamination of mutant RNAs which were recovered as replication competent (for an example, see Fig. 7).
Summary of experimental results obtained during five independent transcription-transfection experiments with each of the diverse DI9c ORF mutants in BHK-21 and MDBK cells. −, replication deficient; +, replication capable. Except for mutant 19 (indicated as +/−), which was found to replicate more efficient in MDBK cells than in BHK cells (see text and Fig. 2), both cell types yielded congruent results.
Summary of five independent in vitro translation-processing experiments performed with each of the diverse DI9c derivatives as templates: “a” indicates that processing of the DI9c mutant-encoded polyprotein was significantly affected with respect to the cleavage profile obtained with the wild-type replicon; “wt” indicates that the mutation exhibited negligible effects on polyprotein cleavage. The overall translation efficiencies of the DI9c ORF mutants did not differ from that of the wild-type RNA (see text and Fig. 3).
Summary of the complementation analysis performed as described in the text. −, not complementable; +, complementable.