Activation of uDNA gene expression by coinfection with integrating virus. A. Upper panels: 2 × 10^5 Jurkat cells were left uninfected (left panel) or infected with increasing amounts of WT-DsRedX virus (5, 20, 60 ng p24 respectively). Lower panels: Cells were simultaneously infected with equal amounts of D116N-GFP virus (10 ng p24) and the same amount of WT-DsRedX virus as in the panel directly above. Numbers represent the percentage of cells in each indicated quadrant. All viruses were envelope-defective and pseudotyped with VSV-G protein to limit viruses to a single round of replication. B. The solid blue line plots the percentage of cells that express D116N-associated GFP fluorescence from the lower panels in A (Y axis) as a function of the amount of WT-DsRedX infection (X axis). Extrapolation to 100% infection with WT-DsRedX virus implies that 42% of cells are infected with a D116N-GFP virus that is capable of generating fluorescence in the presence of a coinfecting integrating virus (dashed lines). C. Effect of Tat on the percentage of cells expressing D116N-associated GFP fluorescence. As in Figure 2A-B, viruses were envelope defective and pseudotyped with VSV-G to overcome differences in CD4 levels on Jurkat and Jurkat-Tat cells and to limit viruses to a single round of replication. qPCR for HIV-1 DNA found equal infection of Jurkat and Jurkat-Tat cells with 0.9 HIV-1 genomes/cell by real time DNA qPCR. A nearly 7-fold increase in the percentage of cells expressing D116N-associated GFP fluorescence is similar to results of coinfection in panel B and demonstrates that Tat is sufficient for the transactivation provided by coinfecting viruses. This experiment is representative of multiple independent experiments. D. The increase in mean fluorescence in Jurkat from D116N-GFP virus as a result of coinfection with an integrase-WT virus as a result of Tat transactivation in the Jurkat-Tat cell line. Coinfection data represent the mean fluorescence of cells coinfected with D116N-GFP and WT-DsRedX viruses divided by the mean fluorescence of cells infected with only D116N-GFP virus. Data represent multiple samples from each of 3 independent experiments. Tat data represent the mean fluorescence of GFP+ Jurkat-Tat cells divided by the mean fluorescence of GFP+ Jurkat cells infected with D116N-GFP virus. The average and SD are derived from multiple samples of a representative experiment.