Placement of Leucine Zipper Motifs at the Carboxyl Terminus of HIV-1 Protease Significantly Reduces Virion Production
Figure 5
Effects of LZ domain insertion on Gag cleavage.
(A) Schematic representations of HIV-1 mutants. Indicated are domains for HIV-1 Gag and pol-encoded p6pol, PR, and RT. Wild-type (Wz) and mutant (Kz) leucine zipper domains and “x” substitution mutations blocking Gag assembly are indicated as described above. (B–C) 293T cells were transfected with designated constructs. At 4 h post-transfection, equal amounts of cells were placed on two dish plates. Panel B: Cells were collected 24 and 48 h post-transfection and subjected to Western immunoblotting. Cellular Pr55gag and p24gag levels were quantified by scanning immunoblot band densities. Ratios of p24gag to p55gag were determined for each mutant and normalized to those of wt in parallel experiments. Bars indicate standard deviation. **, p<0.01. Panel C: Supernatants and cells were collected 48–72 h post-transfection following treatment with or without 5 µM of HIV-1 protease inhibitor Saquinavier. Samples were prepared and subjected to Western immunoblotting.