Figure 5

Comparison of titers produced by PR expression plasmids: wt versus T26S mutant. NIH 3T3 cells were infected with serial dilutions of viral supernatants produced by the 7 plasmid system with either the wt (blue) or T26S mutant (red) PR. Titers were determined by monitoring transduced NIH 3T3 cells for the production of GFP by FACS. In this study, the lentiviral vector (wt-LTR expressing GFP), Rev, Tat, VSV-G, Gag (non-optimized), and Vpr-RT/IN DNA amounts remained constant, while the DNA amounts of Vpr-wt PR (wt protease, shown in blue) and Vpr-T26S PR (mutant protease, shown in red) varied. Experiments were performed using two concentrations for Gag and Vpr-RT/IN: (1×) using 1.3 μg Gag and 2.3 μg Vpr-RT/IN DNA with varying amounts of PR DNA (0.7 μg, 1.0 μg, 1.3 μg, and 1.6 μg), indicated on the graph by circles (●), and (2×) using 2.6 μg Gag and 4.5 μg Vpr-RT/IN DNA along with varying amounts of PR DNA (0.8 μg, 1.4 μg, 2.0 μg 2.6 μg, 3.2 μg), indicated on the graph by triangles (▲). In these initial studies the Vpr-RT/IN plasmid did not contain Vif, the functional titers ranged from 0.4 × 10⁵ TU/ml to 3.0 × 10⁵ TU/ml. N = 1.