Although the RT domain is essential for proper PR-mediated Gag cleavage, the RT homodimerization domain has no enhancement effect on Gag cleavage [48] unless EFV (an HIV-1 RT-dimerization enhancer) is added to culture medium [23, 49] . In contrast, we found that LZ is capable of triggering Gag cleavage enhancement when placed at the PR C-terminus. The RT domain apparently plays a role in preventing premature PR activation. Previous studies suggest that the downstream structural conformation of PR is important in terms of modulating PR activation. First, a single RT amino acid substitution (W402A) that is not known to have major impacts on in vitro RT dimerization [50] markedly enhances Gag processing [31] . An additional partial deletion at the C-terminus of p66RT not only reverses W402Atriggered Gag cleavage enhancement, but also markedly impairs virus maturation. In contrast, truncated Gag-Pol mutants that retain intact p66 or p51 RT domains still respond to the enhancement effect of W402A on Gag processing [31] . Second, substitution mutations in RT are capable of neutralizing the enhancement effect of EFV on Gag processing [49] . Combined, these data suggest that conformational changes in Gag-Pol may significantly affect PR dimer interface interactions, leading to either premature or insufficient PR activation. Our finding that the fusion of HIV-1-unrelated dimerization protein sequences at the C-terminus of PR sharply enhanced Gag cleavage strongly supports the hypothesis that structural conformation, rather than specific sequences, largely determines PR activation status. The RT structure domain in the Gag-Pol context may help prevent PR from premature activation via a conformation that prevents the Gag and Gag-LZ chimeras. HIV-1 Gag domains, pol-encoded PR, and the transframe peptide p6 pol are indicated; fs denotes a frameshift mutation that forces gag and pol into the same reading frame. The fused leucine zipper (LZ) domains wt (Wz) or mutant (Kz) at the PR C-terminus are indicated as described in the Figure 1 legend; x denotes substitution mutations in CA, NC (NC15A), and PR that blocked either Gag assembly or PR activity. (B-E) Assembly and multimerization of HIV-1 Gag mutants. 293T cells were transfected with designated constructs. At 48-72 h post-transfection, culture supernatants and cells were collected and subjected to Western immunoblotting (panels B and C). (D) Gag-associated proteins from medium or cell samples were quantified by scanning immunoblot band densities (C). Ratios of Gag in media to Gag in cells were determined for each construct, and compared with wt release level by dividing the release ratio for each chimera by the wt ratio. (E) Velocity sedimentation analysis of cytoplasmic Gag precursor and Gag-LZ chimeras. Homogenized and extracted cytoplasmic lysates were centrifuged through consecutive 25%, 35%, and 45% sucrose gradients at 130,0006g for 1 hour. Fractions were collected from the top of each gradient. Aliquots of each fraction were subjected to SDS-PAGE (10%) and probed with a monoclonal antibody directed against HIV-1 CA. doi:10.1371/journal.pone.0032845.g004 PR dimer interface from interacting efficiently. This conformation may change during Gag-Pol packaging, thus supporting more efficient PR dimer interaction.