Abstract
BACKGROUND: 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 ((mdr)PRs). This study aimed to develop a fission yeast cell-based system for rapid testing of new PIs that combat (mdr)PRs. RESULTS: Three (mdr)PRs were isolated from HIV-infected patients that carried seven ((M7)PR), ten ((M10)PR) and eleven ((M11)PR) 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 (mdr)PRs 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 ((wt)PR) and the (M7)PR-mediated activities. However, none of them were able to suppress the (M10)PR or the (M11)PR. CONCLUSIONS: The three clinically isolated (mdr)PRs maintained their viral proteolytic activities and drug resistance in the fission yeast. Furthermore, those viral (mdr)PR 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 (wt)PR in fission yeast as they did in mammalian cells. Significantly, two of the high level (mdr)PRs ((M10)PR and (M11)PR) were resistant to all of the existing PI drugs including DRV. This observation underscores the importance of continued searching for new PIs against (mdr)PRs.