A fission yeast cell-based system for multidrug resistant HIV-1 proteases

HIV-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.

Three _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.

The 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.