Figure 6.

Blocking PR-RT cleavage significantly enhances G2AP virus titers. (A) Schematic representations of HIV-1 Gag and Gag–Pol expression constructs. HIV-1 Gag protein domains and pol-encoded proteins are indicated as described in the Figure 1 caption. F99V denotes blocking mutation at the PR/RT site, with the Phe residue at the PR C-terminus replaced with Val. (B,C) HEK 293T cells were transfected with designated constructs. Panel B: At 4 h post-transfection, equal amounts of cells were plated on three dishes and either left untreated, treated with the HIV-1 protease inhibitor saquinavir (lanes 3 and 6), or with the RT-dimerization enhancer efavirenz (lanes 4 and 7). Cells and supernatants were collected 48 h post-transfection and analyzed by Western immunoblotting. Membrane-bound proteins were probed with anti-p24CA and anti-RT antibodies. Asterisks indicate positions of PR linked with p66RT. Virus assembly efficiency (D) for each mutant was determined as described in the Figure 5 caption. Error bars indicate standard deviation. ** p < 0.01; *** p < 0.001.