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

We looked for correlations between enhanced PR-mediated Pr55 gag cleavage efficiency and increased Gag-PR-LZ multimerization capacity. Believing that the potent Gag assembly domain might determine chimera multimerization status, we predicted that the contribution of LZ to enhanced chimera multimerization would be barely (if at all) detectable. We therefore assessed chimera multimerization capacity in a Gag assemblydefective context. After constructing an assembly-defective mutant (designated MoGag) and confirming that the mutation significant-ly impaired Gag assembly (Fig. 4B ), we cloned PR-LZ chimeras into MoGag. To block the effect of PR activity on Gag-PR-LZ chimera assembly assays, all chimeras were introduced into a PRinactivated HIV-1 Pr160 gag-pol -expression plasmid GPfs [4] , with gag and pol in the same reading frame (Fig. 4A ). Results from repeated independent experiments indicate that chimeras with predicted molecular weights were detected in both supernatants and cell lysates following transient expression in 293T cells, suggesting that all chimeras were capable of assembly and release to some extent. However, we noted that MoGagfsWz transfectants produced more chimeric VLPs than MoGagfsD or MoGagfsKz (Figs. 4C and 4D). To confirm that MoGag was multimerizationdefective and that LZ did enhance assembly-defective Gag multimerization, we subjected wt Gag and each mutant to velocity sedimentation analyses. A non-myristylated (myr-) Gag mutant [32] known to be severely defective in both membrane binding and multimerization served as a negative control. Our data indicate that most of the wt Gag was recovered at fractions 3 to 5; in contrast, most myr-Gag and substantial amounts of MoGag were recovered at fractions 1 and 2. Portions of MoGagfsD and MoGagfsKz were detected at lower sucrose density fractions, whereas MoGagfsWz was almost completely recovered at higher sucrose density fractions (Fig. 4E ). Unlike MoGagfsWz, which mostly sedimented at fractions 4 and 5, considerable amounts of MoGagfsWKz and MoGagfsKWz were also recovered at fraction 3, and low but detectable amounts were observed in fraction 2. This sedimentation pattern was similar to that of MoGagfsKz (Fig. 4E , three bottom panels). Also similar to MoGagfsKz, both MoGagfsWKz and MoGagfsKWz were incapable of efficiently assembling into chimeric VLPs (data not shown). We observed this result in repeated independent experiments. This finding suggests that when fused to the PR C-terminus, a LZ tandem repeat containing a wt and mutant LZ (WKz or KWz) does not enhance Gag-PR multimerization as effectively as a wt LZ tandem repeat (Wz). This may partly explain why PRWKz and PRKWz showed relatively lower Gag cleavage efficiency compared to PRWWz (Fig. 2) .