Figure 5

Measurement of the efficiency of uDNA replication during coinfection. A. The relationship between the infection of producer cells with WT-DsRedX virus and the presence of producer cells displaying fluorescence from both WT-DsRedX and D116N-GFP viruses. The same amount of D116N-GFP virus was used to infect each population of cells, yielding 4.5% GFP+ cells without addition of WT-DsRedX virus. As increasing amounts of WT-DsRedX virus are used to coinfect cells, the percentage of cells displaying both GFP and DsRedX fluorescence increased linearly, as shown. Data were collected day 2 after infection of producer cells, at the time of virus transfer to target cells. Values represent the percentage of producers that were double-positive (X axis) vs. the percentage of producers that were DsRedX+ (both single and double positive). B. The relationship between the frequency of double positive GFP+DsRedX+ producer cells and the frequency of the resulting GFP+ target cells. C. Relationship between the GFP+ producer cells and the ratio of D116N-GFP and WT-DsRedX viruses conferred to target cells. The X axis predicts the percentage of viruses generated by producer cells that will confer GFP to target cells. The formula for the X axis assumes equal production of D116N-GFP and WT-DsRedX viruses from double positive producer cells and production of only DsRedX viruses from DsRedX+ cells. The Y axis presents the percentage of all fluorescent target cells that are GFP+. In the target cells, all GFP+ cells (G) and DsRedX+ cells (R) are tallied, and cells which are double positive GFP+DsRedX+ are counted in both categories. The red line represents unity between the two formulas, where the assumptions used in the X axis formula are true. D. The experiment presented in A-C was repeated using primary activated CD4+ T cells as producers, and the percentage of D116N-GFP and WT-DsRedX in each producer cell population is shown to illustrate the wide range of MOI and WT/D116N ratios employed. Blue squares represent producer cells on day 2 after infection and orange circles represent producer cells on day 3 after infection. Data are aggregated from 3 independent experiments. E. The relationship between producer and target cells as in Figure 5C using primary T cell producer cells, showing data from the 3 independent experiments in Figure 5D. F. WT-DsRedX to D116N-GFP ratio in the target cells by flow cytometry and DNA PCR, and by RT-PCR on the viruses used to infect them. Numbers represent the ratio of DsRedX+ cells to GFP+ cells, or the ratio of WT-DsRedX to D116N-GFP nucleic acid in indicated samples. This experiment is representative of two independent experiments. G. Averages and standard deviations for the cumulative data in Figure 5E.