Abstract:BackgroundHIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR wi… Show more
“…Our prior results have shown that the wt PR 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 wt PR in fission yeast was similar to that in mammalian cells [32]. The experiment conducted here was aimed to test whether the three clinically isolated HIV-1 mdr PRs were also functional as PR enzymes in fission yeast.…”
Section: Resultsmentioning
confidence: 99%
“…Conversely, separation of GFP from Vpr due to the PR cleavage at either the MA↓CA (DSQNY↓PIVQ) or the p6 (DSFNF↓PQIT) substrate site will lead to the “GFP pattern,” i.e., with uniform distribution throughout cells [35]. Note that our early test results showed that the PR-mediated cleavages on the MA↓CA (DSQNY↓PIVQ) and on the p6 (DSFNF↓PQIT) substrates were target specific, and the wt PR by itself did not interfere with the intracellular localization of GFP or GFP-tagged Vpr in the fission yeast [32]. Therefore, we were able to measure the specific enzymatic activities of HIV-1 mdr PR as designed.Fig.…”
Section: Resultsmentioning
confidence: 99%
“…1B). When IDV was added to the wt PR-producing cells, it prevented the wt PR protein cleavage activities [32]. This inhibitory activity was reflected by the reversion of the “GFP pattern” back to the “Vpr pattern”.…”
Section: Resultsmentioning
confidence: 99%
“…This HTS platform was later adapted by the Molecular Libraries Probe Production Centers Network in the Molecular Libraries Program at NIH. Most recently, we showed for the first time that the wild type HIV-1 PR ( wt PR) proteolyzes the HIV-1 viral substrates in fission yeast in the same manner as it does in the mammalian cells [32, 33]. As a follow-up of this initial finding, in this study, our primary goal was to develop a fission yeast cell-based system for functional analysis of HIV-1 mdr PRs.…”
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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-016-0131-5) contains supplementary material, which is available to authorized users.
“…Our prior results have shown that the wt PR 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 wt PR in fission yeast was similar to that in mammalian cells [32]. The experiment conducted here was aimed to test whether the three clinically isolated HIV-1 mdr PRs were also functional as PR enzymes in fission yeast.…”
Section: Resultsmentioning
confidence: 99%
“…Conversely, separation of GFP from Vpr due to the PR cleavage at either the MA↓CA (DSQNY↓PIVQ) or the p6 (DSFNF↓PQIT) substrate site will lead to the “GFP pattern,” i.e., with uniform distribution throughout cells [35]. Note that our early test results showed that the PR-mediated cleavages on the MA↓CA (DSQNY↓PIVQ) and on the p6 (DSFNF↓PQIT) substrates were target specific, and the wt PR by itself did not interfere with the intracellular localization of GFP or GFP-tagged Vpr in the fission yeast [32]. Therefore, we were able to measure the specific enzymatic activities of HIV-1 mdr PR as designed.Fig.…”
Section: Resultsmentioning
confidence: 99%
“…1B). When IDV was added to the wt PR-producing cells, it prevented the wt PR protein cleavage activities [32]. This inhibitory activity was reflected by the reversion of the “GFP pattern” back to the “Vpr pattern”.…”
Section: Resultsmentioning
confidence: 99%
“…This HTS platform was later adapted by the Molecular Libraries Probe Production Centers Network in the Molecular Libraries Program at NIH. Most recently, we showed for the first time that the wild type HIV-1 PR ( wt PR) proteolyzes the HIV-1 viral substrates in fission yeast in the same manner as it does in the mammalian cells [32, 33]. As a follow-up of this initial finding, in this study, our primary goal was to develop a fission yeast cell-based system for functional analysis of HIV-1 mdr PRs.…”
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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-016-0131-5) contains supplementary material, which is available to authorized users.
“…Cell viability was evaluated by Trypan blue staining (50) and by a commercial live/dead yeast viability assay (Invitrogen) (47,51). Trypan blue is a diazo dye that stains only dead cells; live cells with intact cell membranes are not stained.…”
The Zika virus (ZIKV) causes microcephaly and the Guillain-Barré syndrome. Little is known about how ZIKV causes these conditions or which ZIKV viral protein(s) is responsible for the associated ZIKVinduced cytopathic effects, including cell hypertrophy, growth restriction, cell-cycle dysregulation, and cell death. We used fission yeast for the rapid, global functional analysis of the ZIKV genome. All 14 proteins or small peptides were produced under an inducible promoter, and we measured the intracellular localization and the specific effects on ZIKV-associated cytopathic activities of each protein. The subcellular localization of each ZIKV protein was in overall agreement with its predicted protein structure. Five structural and two nonstructural ZIKV proteins showed various levels of cytopathic effects. The expression of these ZIKV proteins restricted cell proliferation, induced hypertrophy, or triggered cellular oxidative stress leading to cell death. The expression of premembrane protein (prM) resulted in cell-cycle G1 accumulation, whereas membrane-anchored capsid (anaC), membrane protein (M), envelope protein (E), and nonstructural protein 4A (NS4A) caused cell-cycle G2/M accumulation. A mechanistic study revealed that NS4A-induced cellular hypertrophy and growth restriction were mediated specifically through the target of rapamycin (TOR) cellular stress pathway involving Tor1 and type 2A phosphatase activator Tip41. These findings should provide a reference for future research on the prevention and treatment of ZIKV diseases. T he recent Zika virus (ZIKV) outbreak was surprising in its rapidity and alarming in its association with microcephaly in newborns (1-3) and the Guillain-Barré syndrome in adults (4, 5). Currently, little is known about ZIKV pathogenicity. ZIKV infects various neuronal cells and affects brain neurogenesis by reducing cellular proliferation and inducing cell-cycle abnormalities, cell death/apoptosis, and autophagy (2, 3, 6-8). However, it is not known which viral determinant(s) cause these cytopathic effects. The objective of this study was to identify ZIKV factors responsible for the ZIKV-mediated cytopathic effects that underlie the diseases associated with ZIKV.
Fission yeast is a single-cell eukaryote that has been used extensively as a model organism to study cell biology and virology of higher eukaryotes including plants and humans. In particular, it is a very well-tested model to study evolutionary highly conserved cellular activities such as cell proliferation, cell cycle regulation, and cell death. Some of the advantages of using fission yeast as a surrogate system: easy to carry out functional and genome-wide analysis of small viral genome, easy to maintain in the laboratory with a relatively short doubling time. It is genetically amendable and can be used to test the effect of gain-of-function or loss-of-function of a gene product. Here, we describe a streamlined and large-scale molecular cloning strategy for genome-wide characterization of small viruses in fission yeast.
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