Despite a lack of direct evidence, it is generally accepted that Gag-Pol molecule dimerization or multimerization triggers HIV-1 PR activation, which mediates Gag and Gag-Pol cleavage. Here we demonstrated that the insertion of LZ at the C-terminus of PR triggers markedly enhanced PR-mediated Gag cleavage efficiency, which is associated with increased Gag-PR multimerization capacity. This suggests that an HIV-1-unrelated protein sequence capable of self-association can enhance Gag cleavage efficiency when fused to the PR C-terminus. It also provides evidence in support of the assumption that Gag-Pol/Gag-Pol interaction triggers PR activation.