Figure 8.

16.4.1 influences transactivation capacity of Rev in human cells. Panel (A) depicts the reduction of Rev activity obtained by cotransfection of 0.5 μg plasmids directing expression of 16.4.1 fusion proteins and 0.1 μg rev expression plasmids. The graph shows mean values and standard deviations of 5 independent experiments carried out in 293T cells expressing either IgG1-16.4.1 or 16.4.1-GFP in combination with Rev-GFP or Rev-CFP. FACS analysis was used to quantify the number of cells expressing the Rev-dependent RFP reporter encoded by pLRed(INS)₂R (see Material and Methods) within the population of transfected cells. The dot plots above the bars (FL1 = green fluorescence, FL3 = red fluorescence) represent examples for evaluation of FACS data for fully functional Rev (left), inhibited Rev function by 16.4.1 coexpression (middle) and background expression of the reporter in the absence of Rev (right). The non-transfected, fluorescence negative 293T cell population is represented in grey and excluded from analyses. 293T cells containing the inactive reporter gene (no red fluorescence) and expressing GFP as transfection control were used to define the transfected cell population (R2, right dot plot). Cells transfected with all plasmids required for activation of reporter gene expression (expression plasmids encoding Rev, Tat and the reporter RFP) as well as for expression of the GFP transfection control contain a population of cells expressing both green and red fluorescent proteins defined as R3. The ratio of R3 to R2 represents the proportion of transfected cells showing Rev-dependent reporter expression (i.e. Rev activity). Rev activity in the absence of 16.4.1 was set at 100% (left bar of the graph). Coexpression of 16.4.1 fusion proteins diminished Rev activity to approximately 50% (middle bar). (B) The graph shows the result of a single experiment analysing the effect of different amounts of 16.4.1-GFP expression on Rev-CFP activity. Rev-CFP activity in the absence of 16.4.1-GFP was set at 100%. Coexpression of different amounts of 16.4.1-GFP reduced Rev activity to 35.1% (0.5 μg pC16.4.1sg143) or 12.8% (1 μg pC16.4.1sg143) demonstrating a dose dependent effect of the 16.4.1-GFP on Rev activity.