Abstract
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.
Highlights
Human immunodeficiency virus type 1 (HIV-1) gag encodes a polypeptide Pr55gag that can self-assemble into virus-like particles (VLPs) [1]
Since enhanced or premature Gag cleavage by PR can lead to significantly reduced virus release, and since the wt leucine zipper (LZ) fusion-containing constructs exhibited higher ratios of cellular p24gag to Pr55gag compared to those found in Gag/PR cell lysates (Fig. 2A, lanes 3–4 vs. lane 2), we suggest that the LZ-associated virus production defect was largely due to enhanced Pr55gag cleavage efficiency
Despite a lack of direct evidence, it is generally accepted that Gag-Pol molecule dimerization or multimerization triggers HIV-1 PR activation, which mediates Gag and Gag-Pol cleavage
Summary
Human immunodeficiency virus type 1 (HIV-1) gag encodes a polypeptide Pr55gag that can self-assemble into virus-like particles (VLPs) [1]. During or soon after virus release from cells, Pr55gag is cleaved by viral protease (PR) into four major products: matrix (MA, p17), capsid (CA, p24), nucleocapsid (NC, p7), and p6 domains [1]. Pr160gag-pol is incorporated into virions via interactions with assembling Pr55gag [3,4,5,6,7,8]. Pr160gag-pol cleavage by PR yields reverse transcriptase (RT) and integrase (IN) in addition to Gag products. The PRmediated proteolytic cleavage of Pr55gag and Pr160gag-pol, known as virus maturation, is essential for the acquisition of viral infectivity [9,10,11,12,13]
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