Fig. 2

Effects of C-terminal p6* amino acid substitutions on protease maturation and virus processing. a Schematic representations of HIV-1 Gag and Gag-Pol expression constructs. HIV-1 Gag protein domains and pol-encoded proteins and the leucine zipper (LZ) motif (striped box, Wz) are indicated as described in the Fig. 1 caption. Also indicated are amino acid residues in the junction area. Two or four C-terminal p6* residues remaining at the LZ/PR junction are shown in boldface. Amino acid changes at the C-terminal p6* tetra-peptide are underlined. Altered or additional residues are in italics. b, c 293T cells were transfected with designated constructs. Culture supernatants and cells were collected at 48–72 h post-transfection. To detect PR-associated products, aliquots of supernatant samples were separated by 4–12% Bis–Tris gradient gels. Membrane-bound proteins were initially probed with anti-PR serum prior to stripping and probing with anti-RT serum, followed by probing with an anti-p24CA monoclonal antibody. Molecular weight size markers (in kDa) are indicated on right side (middle panels). d Relative virus particle processing efficiency of HIV-1 mutants. Virus-associated 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 the wt in parallel experiments. Bars indicate standard deviations. *p < 0.05; **p < 0.01; ***p < 0.001