TABLE 1.
RNA sequence composition of particles released from the two-plasmid system
| Cell line and Gag-Pol proportion (%)a | % RNA sequence identity by strainb |
|||
|---|---|---|---|---|
| Wild type | S1d mutant | CAG mutant | Other | |
| 293T | ||||
| Wild type | 100 | 0 | 0 | 0 |
| 0 | 0 | 100 (5.00) | 0 | 0 |
| 20 | 0 | 81.25 (3.12) | 18.75 | 0 |
| 40 | 0 | 77.77 (16.6) | 22.23 | 0 |
| 60 | 0 | 47.36 (5.26) | 47.36 | 5.28 |
| 80 | 0 | 52.63 | 47.37 | 0 |
| 100 | 0 | 0 | 100 (5.55) | 0 |
| Rat2 | ||||
| Wild type | 100 | 0 | 0 | 0 |
| 0 | 0 | 100 (11.76) | 0 | 0 |
| 20 | 0 | 100 (8.33) | 0 | 0 |
| 40 | 0 | 92.30 | 0 | 7.70 |
| 60 | 0 | 91.66 (16.66) | 8.34 | 0 |
| 80 | 66.66 | 33.34 (11.11) | 0 | 0 |
293T cells were transfected with different proportions of pNCA-S1d (Gag only, represented by 0% Gag-Pol) and/or pNCA-CAG (Gag-Pol only) providing an effective readthrough efficiency from 0 to 100%, and released virus was used subsequently to infect Rat2 cells. The culture medium was sterile filtered, and viral RNA was isolated and reverse transcribed using avian myeloblastosis virus RT. The resulting cDNA was amplified by PCR. PCR amplification products were cloned into the pGEM-T-Easy vector (Promega), and different clones were sequenced from 293T-transfected cells and Rat2-infected cells.
The numbers in parentheses show the percentage of the respective sample that contains one or more point mutations.