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

To determine whether forced PR dimer interactions affect virus assembly and processing, we fused a LZ protein dimerization domain either singly or in tandem repeat to the C-terminus of an HIV-1 Gag-Pol truncated construct (Gag/PR), which is virusassembly competent but processing-defective [23] . The resulting constructs were designated PRWz and PRWWz (Fig. 1) . We used PRKz and PRKKz constructs containing the dimerizationdefective LZ mutant version (Kz) as controls. Kz fusion to PRWz at the wt LZ C-and N-termini yielded constructs PRWKz and PRKWz, respectively. Each mutant was transiently expressed in 293T cells. Virus particle assembly and processing were analyzed by Western immunoblotting. The results shown in Figure 2A indicate that Gag/PR transfectants produced substantial quantities of VLPs at levels that were near wild-type. Unprocessed Gag (the Gag precursor Pr55) and incompletely processed Gag (the intermediate p41gag) represent two major Gag products compared to wt in our supernatant and cell samples ( Fig. 2A , lanes 1 vs. 2). This is consistent with a report stating that a deletion in a downstream pol sequence significantly impairs PR-mediated virus maturation [23] .