Figure 3

The 7 plasmid system: functional titers and modifications. (A) Functional titers were obtained using a wt-LTR lentiviral vector containing green fluorescent protein (GFP) driven by an EF1α promoter. NIH 3T3 cells were infected with serial dilutions of viral supernatants produced by the 5, 6, or 7 plasmid systems as mentioned in the Methods. The number of transducing units (TU) was determined by multiplying the number of cells plated by the percentage of GFP positive cells (determined by FACS) by the dilution factor. The mean titer for the 5 plasmid system, shown in blue, was 2.2 × 10⁶ TU/ml, for the 6 plasmid system, shown in green, 2.4 × 10⁵ TU/ml, for the 7 plasmid system with the optimized Gag, shown in light pink, 4.4 × 10⁵ TU/ml, and for the 7 plasmid system, shown in dark pink, 7.4 × 10⁵ TU/ml. Error bars represent SEM, 5 independent experiments are represented (N = 5), * p = 0.03 (6P versus 7P-Opt), ** p = 0.003 (7P-Opt versus 7P), *** p < or = 0.0002 (6P versus 7P, 5P versus 6P, and 5P versus 7P-Opt, 5P versus 7P) as determined by unpaired t-test using Prism 4 software. (B) Schematic showing the safety modifications incorporated into the 7 plasmid system. Gag proteins are represented in blue and Pol proteins in green. (1) The Gag to Gag-Pol frameshift was eliminated (AAT TT TTA GGG became AAC TTC TTA GGG). (2) PR was expressed independently of both Gag and Pol. In addition, the active site of PR was changed from DTG to DSG to create the T26S mutant PR. (3) The sequences that overlapped between the packaging signal (Ψ) and Gag, and between Gag and Pol (at P6) were greatly reduced.