The Pearson product-moment correlation coefficient was generated to measure the correlation between viral titer and viral RNA copy number in CW sample using GraphPad Prism 5.0 (GraphPad Software, Inc.). Statistical significance for all other experiments was determined using Student's t test. Placement of Leucine Zipper Motifs at the Carboxyl Terminus of HIV-1 Protease Significantly Reduces Virion Production Natural HIV-1 protease (PR) is homodimeric. Some researchers believe that interactions between HIV-1 Gag-Pol molecules trigger the activation of embedded PR (which mediates Gag and Gag-Pol cleavage), and that Gag-Pol assembly domains outside of PR may contribute to PR activation by influencing PR dimer interaction in a Gag-Pol context. To determine if the enhancement of PR dimer interaction facilitates PR activation, we placed single or tandem repeat leucine zippers (LZ) at the PR C-terminus, and looked for a correlation between enhanced Gag processing efficiency and increased Gag-PR-LZ multimerization capacity. We found significant reductions in virus-like particles (VLPs) produced by HIV-1 mutants, with LZ fused to the end of PR as a result of enhanced Gag cleavage efficiency. Since VLP production can be restored to wt levels following PR activity inhibition, this assembly defect is considered PR activity-dependent. We also found a correlation between the LZ enhancement effect on Gag cleavage and enhanced Gag-PR multimerization. The results suggest that PR dimer interactions facilitated by forced Gag-PR multimerization lead to premature Gag cleavage, likely a result of premature PR activation. Our conclusion is that placement of a heterologous dimerization domain downstream of PR enhances PR-mediated Gag cleavage efficiency, implying that structural conformation, rather than the primary sequence outside of PR, is a major determinant of HIV-1 PR activation. Human immunodeficiency virus type 1 (HIV-1) gag encodes a polypeptide Pr55 gag that can self-assemble into virus-like particles (VLPs) [1] . During or soon after virus release from cells, Pr55 gag is cleaved by viral protease (PR) into four major products: matrix (MA, p17), capsid (CA, p24), nucleocapsid (NC, p7), and p6 domains [1] . PR is encoded by pol, which is initially translated as a Pr160 gag-pol polyprotein by a ribosomal frameshift event that occurs at a frequency of 5%, resulting in the expression of Pr160 gag-pol to Pr55 gag at a ratio of approximately 1:20 [2] . Pr160 gag-pol is incorporated into virions via interactions with assembling Pr55 gag [3, 4, 5, 6, 7, 8] . Pr160 gag-pol cleavage by PR yields reverse transcriptase (RT) and integrase (IN) in addition to Gag products. The PRmediated proteolytic cleavage of Pr55 gag and Pr160 gag-pol , known as virus maturation, is essential for the acquisition of viral infectivity [9, 10, 11, 12, 13] .