TABLE 3.
Complementation of defective RNA transcripts from pMTΔ417 and pMTΔ999
| Parameter | RNA at times post-transfectiona
|
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| pMTΔ417 + pMTΔ999
|
pMTΔ417
|
pMTΔ999
|
||||||||
| 0 h | 2 h | 4 h | 16 h | 24 h | 36 h | 72 h | 5 days | 5 days | 5 days | |
| Cytopathologyb | − | − | − | − | − | − | − | + | − | − |
| RT-PCR of RNA from trans- fection supernatantsc | Δ417, Δ999 | Δ417, Δ999 | Δ417, Δ999 | Δ417, Δ999 | Δ417, Δ999 | Δ417, Δ999 | Δ417, Δ999 | Δ417, Δ999 | Δ417 | Δ999 |
| Infectiond | − | − | − | − | − | − | + | + | − | − |
| RT-PCR of RNA from infection supernatantse | ND | ND | ND | ND | ND | ND | Δ417, Δ999 | Δ417, Δ999, stf | ND | ND |
Construction of plasmids containing genomes with deletions Δ417 and Δ999 (pMTΔ417 and pMTΔ999, respectively), in vitro transcription, and BHK-21 cell transfection are described in Materials and Methods. Boldface type indicates the presence of complementation of defective RNAs.
Cytopathology of 106 cells at the indicated times posttransfection; +, >90% cell killing; −, no detectable cytopathology. In transfections with about 500 ng of an RNA transcript of plasmid pMT28 (standard C-S8c1 RNA, described in Materials and Methods) complete cytpathology is observed between 48 and 72 h posttransfection; the equivalent time for O1K transcripts was about 12 h.
The amplifications used were numbers 10, 17, and 21 (Fig. 1A) and 29 (Table 1). At 5 days posttransfection, Δ417 and Δ999 RNA levels in the cotransfection supernatants were about 1.0×104 molecules of RNA per cell, versus 1.4× 104 at time zero after coelectroporation (determined by real-time PCR) (see Materials and Methods). In transfections with Δ999 alone, the amount of RNA was about 7 × 101 molecules per cell at 5 days posttransfection, versus 5×103 molecules per cell at time zero after electroporation.
Infection of 104 BHK-21 cells with the supernatant obtained at different times posttransfection is indicated. +, >90% cell killing; −, no detectable cytopathology.
The amplifications used were numbers 10, 17, and 21 (Fig. 1A) and 29 (Table 1); ND, not determined.
Sequence analysis revealed that the standard (st) RNA probably arose by recombination of Δ417 and Δ999 RNAs (data not shown; see Discussion).