| Summary: | 碩士 === 國立陽明大學 === 臨床醫學研究所 === 99 === The human immunodeficiency virus type 1 (HIV-1) protease (PR) mediates the proteolytic processing of virus particles during or after virus budding. How PR is activated to mediate virion maturation is not completely understood. It is believed that the interaction between Pr160gag-pol molecules triggers activation of embedded PR, which in homodimeric form mediates Gag and Gag-Pol cleavage following its autocleavage from Pr160gag-pol. In addition, the natural reverse transcriptase (RT) is in heterodimeric form, it is speculated that the RT in the Gag-Pol context may facilitate the Pr160gag-pol-Pr160gag-pol interaction via RT-RT interaction and therefore influences the PR activation. Nevertheless, it lacks direct evidence to support that facilitated PR-PR interaction leads to PR activation. We previously study showed that a series of HIV Gag-pol mutants by progressive deletion of the pol sequence downstream of the viral protease (PR) domain which can effects the PR process . Moreover, we have constructed a Gag-Pol deletion mutation involving the reverse transcriptase connection domain almost completely negates the enhancement effect of efavirenz (a nonnucleoside reverse transcriptase inhibitor) on PR-mediated virus processing efficiency. These data suggest that the sequence downstream of the HIV-1 PR may contribute to the PR activation process. To address this issue, we constructed a series of substitution mutation in the PR downstream. We inserted a leucine zipper (LZ) motif at the C-terminus of PR and mutated at the RT thumb domain. Virus-like particles (VLPs) produced by HIV-1 mutants containing LZ fused to the end of PR were significantly reduced as a result of enhanced Gag cleavage efficiency. The assembly defect is PR activity dependant since VLP production can be restored to wt level following inhibition of PR activity. The LZ enhancement effect on Gag cleavage correlated with an enhancement effect on multimerization of Gag-PR molecules. The mutations at RT thumb domain cause unstably RT structure and impair virus particle processing . The results suggest that facilitated PR dimer interactions by forcing Gag-PR multimerization lead to premature Gag cleavage likely subsequent to premature activation of PR.
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