Figure 7.

Measurement of Rev-RRE functional activity from an integrated chromatin associated provirus. HIV- and MSCV-derived constructs as in Fig. 1b were packaged and VSV-G pseudotyped in 293T/17 cells. A single HIV-derived construct with an NL4-3 RRE was used along with three MSCV constructs with 9-G, 8-G, or NL4-3 Rev sequences. CEM-SS cells were transduced with both the HIV-derived construct and one of the MSCV-derived constructs at a multiplicity of infection of 0.5. After 72 hours, fluorescence was measured via flow cytometry and gating for analysis was performed as in Fig. 2e (i.e. analyzed cells fluoresce with mCherry, and with eGFP and/or TagBFP). To generate dot plots, single cells were plotted with Rev-dependent eGFP signal along the y-axis and the Rev-independent TagBFP signal along the x-axis with the (a) NL4-3 Rev, (b) 8-G Rev, and (c) 9-G Rev. Flow cytometry plots represent all mCherry-positive single cells measured in three experimental replicates prior to analysis gating. Plot (a) shows 5,690 cells, (b) shows 12,734 cells, and (c) shows 8,534 cells. (D) Relative Rev-RRE functional activity was calculated for each Rev construct as the ratio of eGFP:TagBFP MFI in the set of cells expressing fluorescent markers from both transducing constructs. The 9-G Rev activity was set as 100 and the other values were normalized accordingly. N = 3, bars represent SEM.