Figure 6.

The effect of inhibitors of Rev function on fluorescent protein expression from the reporter vectors. (a) Compound 103833 decreases Rev-RRE functional activity in a dose dependent manner but does not affect CTE function. Two vector constructs were tested in the presence of compound 103833, a Rev-pathway small molecule inhibitor. The RRE construct was identical to that in Fig. 1a. The CTE construct was created by replacing the RRE with an MPMV CTE. The constructs were transfected into 293T/17 cells with Rev (RRE construct) or Nxf1 and NxT1 (CTE construct). Cells were pre-treated with the indicated concentrations of 103833 for 24 hours before transfection and remained in the medium until 24 hours after transfection when the cells were harvested for analysis. Both eGFP and mCherry signals were quantified for both constructs. The MFI for each fluorescent signal in the absence of inhibitor was set as 100 and the other values were normalized accordingly. (b) Trans-dominant negative Rev M10 inhibits Rev-RRE functional activity in a dose dependent manner. 4 × 10⁵ 293T/17 cells were transfected with 1000 ng of an eGFP-TagBFP HIV-derived vector along with 75 ng of pMSCV-Rev-IRES-mCherry vector (see Fig. 1b). Additionally, a variable mass of a plasmid expressing trans-dominant negative Rev-M10 plasmid (pCMV-TD-Rev) was transfected into the system along with an empty vector to equalize total DNA mass of the pCMV plasmids to 600 ng. The MFI of the Rev-dependent (eGFP), Rev-independent (TagBFP), and Rev-linked (mCherry) signals was determined for each ratio of Rev plasmid to Rev-M10 plasmid. For each fluorescent protein, the MFI in the absence of Rev-M10 was set at 100 and the other values were normalized accordingly. N = 2, bars, which are too small to be seen in this figure, represent SEM for both graphs. MFI = arithmetic mean fluorescence intensity.