Figure 5.

The functional activity of S97A and T174A single and double mutants. (A) The activity of either wtRex-1 or Rex-1 mutants, as indicated, was determined using the modified HIV p24 Gag reporter assay. The specific amino acid substitution for each Rex-1 mutant is shown. Cells were transfected and Rex activity was determined as described in the legend to Figure 4. The values represent actual p24 Gag production from a representative experiment performed in triplicate. Error bars indicate standard deviations. T, threonine; S, serine; A, alanine; D, aspartic acid. Whole cell lysates normalized for transfection efficiency were subjected to Western blot (shown below) using rabbit Rex-1-specific antisera. (B) 293T cells were transfected with 0.25 μg pcTat, 0.5 μg pCgagRxRE-I, 0.05 μg CMV-luc, and 0.1 μg of wtRex-1 or 0.1 μg of wtRex-1 + 0.2 μg of S97A, T174A Rex-1 mutant plasmid. Cell lysates were prepared 24 hours post-transfection and p24 Gag levels were determined by HIV-1 p24 Gag ELISA. Rex-1 functional assay reveals that the double mutant (S97A, T174A) does not inhibit the function of wtRex-1 and thus is not a trans dominant protein.