To determine whether LZ placement affected virion production by wt or assembly-competent mutants in trans, we coexpressed PRWz or PRWWz with the wt or the HIV-1 protease-defective mutant D25, and observed that virus-associated Gag was markedly reduced when D25 was cotransfected with either PRWz or PRWWz at a 1:1 ratio (Fig. 2C ). Similar results were observed when the PRWz or PRWWz was coexpressed with a wt HIV-1 expression vector (data not shown). Combined, these results suggest that (a) PRWz and PRWWz both provided functional PR, and (b) the LZ-triggered virion assembly defect was primarily due to a higher Gag cleavage efficiency. The PRWWz transfectant frequently expressed a lower Gag level compared to other constructs (Fig. 2B, lane 12) , likely the result of increased proteolytic degradation of Gag mediated by the PR.