Figure 5.

PR activity attenuation enhances G2AP virus titers. (A) Schematic representations of HIV-1 Gag and Gag–Pol expression constructs. Indicated are HIV-1 Gag protein domains, pol-encoded proteins, and the 2A peptide as described in the Figure 1 caption. Asterisks denote the T26S mutation, in which Ser is substituted for the Thr26 of PR. (B) HEK 293T cells were transfected with a designated construct. Two days post-transfection, supernatants and cells were collected, prepared, and subjected to Western blot analysis. (C) Virus particle processing efficiency data. Quantities of Pr55gag and mature p24gag were measured by scanning their respective band densities from immunoblots. p24gag-to-Pr55gag quantity ratios were calculated for each construct and compared with wt ratios—that is, the p24gag/Pr55gag ratio for each mutant was divided by the wt p24gag/Pr55gag ratio in parallel experiments. Error bars indicate standard deviations. * p < 0.05; ** p < 0.01; *** p < 0.001. (D) Relative virus release efficiency. Virus-associated or cellular Gag protein levels were quantified by scanning p24gag-associated band densities from immunoblots. Ratios of total virus-associated Gag protein level to total cellular Gag protein level were calculated and compared to the wt by dividing the ratio for each mutant by the ratio for the wt in parallel experiments. Error bars indicate standard deviation. * p < 0.05; ** p < 0.01; *** p < 0.001.