title: A fission yeast cell-based system for multidrug resistant HIV-1 proteases.
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 (mdrPRs). This study aimed to develop a fission yeast cell-based system for rapid testing of new PIs that combatmdrPRs.
  
  
  RESULTS
  ThreemdrPRs were isolated from HIV-infected patients that carried seven (M7PR), ten (M10PR) and eleven (M11PR)PRgene 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 threemdrPRs 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 theM7PR-mediated activities. However, none of them were able to suppress theM10PR or theM11PR.
  
  
  CONCLUSIONS
  The three clinically isolatedmdrPRs maintained their viral proteolytic activities and drug resistance in the fission yeast. Furthermore, those viralmdrPR 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 thewtPR in fission yeast as they did in mammalian cells. Significantly, two of the high levelmdrPRs (M10PR andM11PR) were resistant to all of the existing PI drugs including DRV. This observation underscores the importance of continued searching for new PIs againstmdrPRs.
authors.affiliation: Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201 USA ; Department of Membrane Biochemistry, Institute of Animal Biochemistry and Genetics, Centre of Biosciences, SAS, 84005 Bratislava, Slovakia.
authors.forename: Zsigmond
authors.initials: Z
authors.lastname: Benko
authors.affiliation: Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201 USA.
authors.forename: Dong
authors.initials: D
authors.lastname: Liang
authors.affiliation: Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201 USA.
authors.forename: Ge
authors.initials: G
authors.lastname: Li
authors.affiliation: Children's Memorial Institute for Education and Research, Northwestern University Feinberg School of Medicine, Chicago, IL 10164 USA.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Elder
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, IN 47907 USA.
authors.forename: Anindya
authors.initials: A
authors.lastname: Sarkar
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, IN 47907 USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Takayama
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, IN 47907 USA.
authors.forename: Arun K
authors.initials: AK
authors.lastname: Ghosh
authors.affiliation: Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201 USA ; Department of Microbiology-Immunology, University of Maryland School of Medicine, Baltimore, MD 21201 USA ; Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201 USA ; Children's Memorial Institute for Education and Research, Northwestern University Feinberg School of Medicine, Chicago, IL 10164 USA.
authors.forename: Richard Y
authors.identifier: 0000-0003-3424-2852
authors.initials: RY
authors.lastname: Zhao
keywords: Cell death; Cell proliferation; Fission yeast; HIV-1; Mitochondria; Multidrug resistant proteases; Oxidative stress; Protease inhibitors; Proteolytic cleavage