Abstract
BackgroundHIV-1 protease (PR) is an essential enzyme for viral production. Thus, PR inhibitors (PIs) are the most effective class of anti-HIV drugs. However, the main challenge to the successful use of PI drugs in patient treatment is the emergence of multidrug resistant PRs (mdrPRs). This study aimed to develop a fission yeast cell-based system for rapid testing of new PIs that combat mdrPRs.ResultsThree mdrPRs were isolated from HIV-infected patients that carried seven (M7PR), ten (M10PR) and eleven (M11PR) PR gene mutations, respectively. They were cloned and expressed in fission yeast under an inducible promoter to allow the measurement of PR-specific proteolysis and drug resistance. The results showed that all three mdrPRs maintained their abilities to proteolyze HIV viral substrates (MA↓CA and p6) and to confer drug resistance. Production of these proteins in the fission yeast caused cell growth inhibition, oxidative stress and altered mitochondrial morphologies that led to cell death. Five investigational PIs were used to test the utility of the established yeast system with an FDA-approved PI drug Darunavir (DRV) as control. All six compounds suppressed the wildtype PR (wtPR) and the M7PR-mediated activities. However, none of them were able to suppress the M10PR or the M11PR.ConclusionsThe three clinically isolated mdrPRs maintained their viral proteolytic activities and drug resistance in the fission yeast. Furthermore, those viral mdrPR activities were coupled with the induction of growth inhibition and cell death, which could be used to test the PI activities. Indeed, the five investigational PIs and DRV suppressed the wtPR in fission yeast as they did in mammalian cells. Significantly, two of the high level mdrPRs (M10PR and M11PR) were resistant to all of the existing PI drugs including DRV. This observation underscores the importance of continued searching for new PIs against mdrPRs.
Highlights
human immunodeficiency virus type 1 (HIV-1) protease (PR) is an essential enzyme for viral production
We showed for the first time that the wild type HIV-1 PR proteolyzes the HIV-1 viral substrates in fission yeast in the same manner as it does in the mammalian cells [32, 33]
HIV‐1 multidrug resistant PRs (mdrPRs) cleave the same indigenous viral protein substrates in fission yeast Our prior results have shown that the wildtype PR (wtPR) cleaved the indigenous HIV-1 MA↓CA (DSQNY↓PIVQ) and p6 (DSFNF↓PQIT) viral targets in the fission yeast as it does in the HIV-1 infection of mammalian cells, suggesting that the protease activity of HIV-1 wtPR in fission yeast was similar to that in mammalian cells [32]
Summary
PR inhibitors (PIs) are the most effective class of anti-HIV drugs. The main challenge to the successful use of PI drugs in patient treatment is the emergence of multidrug resistant PRs (mdrPRs). This study aimed to develop a fission yeast cell-based system for rapid testing of new PIs that combat mdrPRs. HIV-1 Protease (PR) is an aspartic protease that normally presents as a homodimer with each subunit consisting of 99 amino acids (12kD) [1]. In spite of the tremendous progress we have made in ARTs, one of the main challenges to the success of ARTs is viral multidrug resistance (MDR) to the anti-HIV drug targets such as PR. The viral multidrug resistant PRs (mdrPRs) are developed primarily due to the continued emergence of viral gene mutations upon prolonged ARTs [10, 11].
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