Placement of Leucine Zipper Motifs at the Carboxyl Terminus of HIV-1 Protease Significantly Reduces Virion Production

Pan, Yen-Yu; Wang, Shiu-Mei; Huang, Kuo-Jung; Chiang, Chien-Cheng; Wang, Chin-Tien

Our next question was whether LZ enhanced the cleavage efficiency of the assembly-defective mutant MoGag. MoGag with an LZ fusion was expressed in a Gag/PR context, thus expressing both Pr55 gag and PR containing Gag-Pol or Gag-PR(-LZ) fusions (Fig. 5A ). As expected, the insertion of LZ into MoGag/PR at the PR C-terminus resulted in significantly enhanced Gag cleavage efficiency (Fig. 5C, lanes 11 vs. 15 ), suggesting that the LZ enhancement of Gag-PR multimerization is correlated with increased PR-mediated Gag cleavage efficiency. Inefficient Gag cleavage, likely due to either impaired PR activation as a result of Pol truncation or to inhibited PR activity due to a protease inhibitor, resulted in improved MoGag VLP assembly (Fig. 5C , 12 and 15-18 ). This suggests that the MoGag assembly defect is PR activity-dependent, at least in part. The MoPRWWz had significantly higher cellular p24 gag /Pr55 gag ratios compared to MoGag/PR, but slightly lower compared to PRWWz (Figs. 5B and 5C), suggesting that the multimerization-defective Gag mutation reduced the LZ-mediated enhancement of Gag cleavage. It also supports the proposal that the Gag assembly domain plays a role in PR activation.