Fig. 1

Effects of C-terminal HIV-1 p6* residue substitutions on virus assembly and processing. a Schematic representations of HIV-1 Gag and Gag-Pol expression constructs. Indicated are the HIV-1 Gag protein domains MA (matrix), CA (capsid), NC (nucleocapsid), p6, pol-encoded p6*, PR, RT and IN. “X” denotes a PR-inactivated mutation. Arrows indicate PR cleavage sites. Underlined “V” indicates a Val residue substitution for the final C-terminal p6* residue Phe. Striped (Wz) box denotes wild-type (wt) leucine zipper (LZ). Remaining C-terminal p6* residues are in boldface. Altered or additional residues are in italics. b Blocking p6*-PR cleavage is insufficient for mitigating the enhanced Gag cleavage incurred by an LZ replacement for a deleted p6* domain. 293T cells were transfected with designated constructs. At 4 h post-transfection, equal amounts of cells were plated on two dishes and either left untreated or treated with saquinavir (a HIV-1 protease inhibitor) at a concentration of 5 μM. Supernatants and cells were collected 48 h post-transfection, prepared, and subjected to Western immunoblotting. c Blocking p6*-PR cleavage (V/P mutation) disrupted the function of a C-terminal p6* tetra-peptide for modulating PR activation. 293T cells were transfected with designated constructs. Culture supernatants and cells were collected and subjected to Western immunoblotting at 48–72 h post-transfection. d Relative virus assembly efficiencies of HIV-1 mutants. Gag proteins from medium or cell samples were quantified by scanning mutant and wt p24gag-associated band densities from immunoblots. Ratios of total Gag protein levels in medium to those in cells were determined for each construct and compared with wt release levels; release ratios for each mutant were divided by wt ratios in parallel experiments. Error bars indicate standard deviation. *p < 0.05; **p < 0.01. e Relative virus particle processing efficiency data for 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