Repression of a large number of genes requires interplay between homologous recombination and HIRA.

During homologous recombination, Dbl2 protein is required for localisation of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments. RNA-seq analysis of dbl2Δ transcriptome showed that the dbl2 deletion results in upregulation of more than 500 loci in Schizosaccharomyces pombe. Compared with the loci with no change in expression, the misregulated loci in dbl2Δ are closer to long terminal and long tandem repeats. Furthermore, the misregulated loci overlap with antisense transcripts, retrotransposons, meiotic genes and genes located in subtelomeric regions. A comparison of the expression profiles revealed that Dbl2 represses the same type of genes as the HIRA histone chaperone complex. Although dbl2 deletion does not alleviate centromeric or telomeric silencing, it suppresses the silencing defect at the outer centromere caused by deletion of hip1 and slm9 genes encoding subunits of the HIRA complex. Moreover, our analyses revealed that cells lacking dbl2 show a slight increase of nucleosomes at transcription start sites and increased levels of methylated histone H3 (H3K9me2) at centromeres, subtelomeres, rDNA regions and long terminal repeats. Finally, we show that other proteins involved in homologous recombination, such as Fbh1, Rad51, Mus81 and Rad54, participate in the same gene repression pathway.

Defining the Functional Interactome of Spliceosome-Associated G-Patch Protein Gpl1 in the Fission Yeast Schizosaccharomyces pombe.

Pre-mRNA splicing plays a fundamental role in securing protein diversity by generating multiple transcript isoforms from a single gene. Recently, it has been shown that specific G-patch domain-containing proteins are critical cofactors involved in the regulation of splicing processes. In this study, using the knock-out strategy, affinity purification and the yeast-two-hybrid assay, we demonstrated that the spliceosome-associated G-patch protein Gpl1 of the fission yeast S. pombe mediates interactions between putative RNA helicase Gih35 (SPAC20H4.09) and WD repeat protein Wdr83, and ensures their binding to the spliceosome. Furthermore, RT-qPCR analysis of the splicing efficiency of deletion mutants indicated that the absence of any of the components of the Gpl1-Gih35-Wdr83 complex leads to defective splicing of fet5 and pwi1, the reference genes whose unspliced isoforms harboring premature stop codons are targeted for degradation by the nonsense-mediated decay (NMD) pathway. Together, our results shed more light on the functional interactome of G-patch protein Gpl1 and revealed that the Gpl1-Gih35-Wdr83 complex plays an important role in the regulation of pre-mRNA splicing in S. pombe.

Relaxation of Some Confusions about Confounders.

This work is about observational causal discovery for deterministic and stochastic dynamic systems. We explore what additional knowledge can be gained by the usage of standard conditional independence tests and if the interacting systems are located in a geodesic space.

Mutations that prevent methylation of cohesin render sensitivity to DNA damage in S. pombe.

The canonical role of cohesin is to mediate sister chromatid cohesion. In addition, cohesin plays important roles in processes such as DNA repair and regulation of gene expression. Mounting evidence suggests that various post-translational modifications, including phosphorylation, acetylation and sumoylation regulate cohesin functions. Our mass spectrometry analysis of cohesin purified from Schizosaccharomyces pombe cells revealed that the cohesin subunit Psm1 is methylated on two evolutionarily conserved lysine residues, K536 and K1200. We found that mutations that prevent methylation of Psm1 K536 and K1200 render sensitivity to DNA-damaging agents and show positive genetic interactions with mutations in genes encoding the Mus81-Eme1 endonuclease. Yeast two-hybrid and co-immunoprecipitation assays showed that there were interactions between subunits of the cohesin and Mus81-Eme1 complexes. We conclude that cohesin is methylated and that mutations that prevent methylation of Psm1 K536 and K1200 show synthetic phenotypes with mutants defective in the homologous recombination DNA repair pathway.

Dbl2 Regulates Rad51 and DNA Joint Molecule Metabolism to Ensure Proper Meiotic Chromosome Segregation.

To identify new proteins required for faithful meiotic chromosome segregation, we screened a Schizosaccharomyces pombe deletion mutant library and found that deletion of the dbl2 gene led to missegregation of chromosomes during meiosis. Analyses of both live and fixed cells showed that dbl2Δ mutant cells frequently failed to segregate homologous chromosomes to opposite poles during meiosis I. Removing Rec12 (Spo11 homolog) to eliminate meiotic DNA double-strand breaks (DSBs) suppressed the segregation defect in dbl2Δ cells, indicating that Dbl2 acts after the initiation of meiotic recombination. Analyses of DSBs and Holliday junctions revealed no significant defect in their formation or processing in dbl2Δ mutant cells, although some Rec12-dependent DNA joint molecules persisted late in meiosis. Failure to segregate chromosomes in the absence of Dbl2 correlated with persistent Rad51 foci, and deletion of rad51 or genes encoding Rad51 mediators also suppressed the segregation defect of dbl2Δ. Formation of foci of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments, was impaired in dbl2Δ cells. Our results suggest that Dbl2 is a novel regulator of Fbh1 and thereby Rad51-dependent DSB repair required for proper meiotic chromosome segregation and viable sex cell formation. The wide conservation of these proteins suggests that our results apply to many species.

Functional characterization of genes encoding cadmium pumping P1B-type ATPases in Aspergillus fumigatus and Aspergillus nidulans.

Several P1B-type ATPases are important Cd2+/Cu2+ pumps in Aspergillus species, and they are tightly associated with the heavy metal stress tolerance of these ascomycetous fungi. To better understand the roles of the two P1B-type ATPases, Aspergillus nidulans CrpA Cd2+/Cu2+ pump (orthologue of the Candida albicans Crp1 Cd2+/Cu2+ pump) and Aspergillus fumigatus PcaA Cd2+ pump (orthologue of the Saccharomyces cerevisiae Pca1 Cd2+ pump), we have generated individual mutants and characterized their heavy metal susceptibilities. The deletion of CrpA in A. nidulans has led to the increased sensitivity of the fungus to stresses induced by Zn2+, Fe2+, or the combination of oxidative-stress-inducing menadione sodium bisulfite and Fe3+. Heterologous expression of A. fumigatus PcaA in the S. cerevisiae pca1 deletion mutant has resulted in enhanced tolerance of the yeast to stresses elicited by Cd2+or Zn2+ but not by Fe2+/Fe3+ or Cu2+. Mammalian host immune defense can attack microbes by secreting Zn2+ or Cu2+, and the oxidative stress induced by host immune systems can also disturb metal (Cu2+, Fe2+, and Zn2+) homeostasis in microbes. In summary, PcaA and CrpA can protect fungal cells from these complex stresses that contribute to the virulence of the pathogenic Aspergillus species. Moreover, due to their presence on the fungal cell surface, these P1B-type ATPases may serve as a novel drug target in the future. IMPORTANCE Mammalian host immune defense disrupts heavy metal homeostasis of fungal pathogens. P1B-type ATPase of Aspergillus fumigatus and Aspergillus nidulans may help to cope with this stress and serve as virulence traits. In our experiments, both A. nidulans Cd2+/Cu2+ pump CrpA and A. fumigatus Cd2+ pump PcaA protected fungal cells from toxic Zn2+, and CrpA also decreased Fe2+ susceptibility most likely indirectly. In addition, CrpA protected cells against the combined stress induced by the oxidative stressor menadione and Fe3+. Since P1B-type ATPases are present on the fungal cell surface, these proteins may serve as a novel drug target in the future.

Model-free detection of unique events in time series.

Recognition of anomalous events is a challenging but critical task in many scientific and industrial fields, especially when the properties of anomalies are unknown. In this paper, we introduce a new anomaly concept called "unicorn" or unique event and present a new, model-free, unsupervised detection algorithm to detect unicorns. The key component of the new algorithm is the Temporal Outlier Factor (TOF) to measure the uniqueness of events in continuous data sets from dynamic systems. The concept of unique events differs significantly from traditional outliers in many aspects: while repetitive outliers are no longer unique events, a unique event is not necessarily an outlier; it does not necessarily fall out from the distribution of normal activity. The performance of our algorithm was examined in recognizing unique events on different types of simulated data sets with anomalies and it was compared with the Local Outlier Factor (LOF) and discord discovery algorithms. TOF had superior performance compared to LOF and discord detection algorithms even in recognizing traditional outliers and it also detected unique events that those did not. The benefits of the unicorn concept and the new detection method were illustrated by example data sets from very different scientific fields. Our algorithm successfully retrieved unique events in those cases where they were already known such as the gravitational waves of a binary black hole merger on LIGO detector data and the signs of respiratory failure on ECG data series. Furthermore, unique events were found on the LIBOR data set of the last 30 years.

Heme Oxygenase-1 (HMX1) Loss of Function Increases the In-Host Fitness of the Saccharomyces 'boulardii' Probiotic Yeast in a Mouse Fungemia Model.

The use of yeast-containing probiotics is on the rise; however, these products occasionally cause fungal infections and possibly even fungemia among susceptible probiotic-treated patients. The incidence of such cases is probably underestimated, which is why it is important to delve deeper into the pathomechanism and the adaptive features of S. 'boulardii'. Here in this study, the potential role of the gene heme oxygenase-1 (HMX1) in probiotic yeast bloodstream-derived infections was studied by generating marker-free HMX1 deletion mutants with CRISPR/Cas9 technology from both commercial and clinical S. 'boulardii' isolates. The six commercial and clinical yeasts used here represented closely related but different genetic backgrounds as revealed by comparative genomic analysis. We compared the wild-type isolates against deletion mutants for their tolerance of iron starvation, hemolytic activity, as well as kidney burden in immunosuppressed BALB/c mice after lateral tail vein injection. Our results reveal that the lack of HMX1 in S. 'boulardii' significantly (p < 0.0001) increases the kidney burden of the mice in most genetic backgrounds, while at the same time causes decreased growth in iron-deprived media in vitro. These findings indicate that even a single-gene loss-of-function mutation can, surprisingly, cause elevated fitness in the host during an opportunistic systemic infection. Our findings indicate that the safety assessment of S. 'boulardii' strains should not only take strain-to-strain variation into account, but also avoid extrapolating in vitro results to in vivo virulence factor determination.

Manifold-adaptive dimension estimation revisited.

Data dimensionality informs us about data complexity and sets limit on the structure of successful signal processing pipelines. In this work we revisit and improve the manifold adaptive Farahmand-Szepesvári-Audibert (FSA) dimension estimator, making it one of the best nearest neighbor-based dimension estimators available. We compute the probability density function of local FSA estimates, if the local manifold density is uniform. Based on the probability density function, we propose to use the median of local estimates as a basic global measure of intrinsic dimensionality, and we demonstrate the advantages of this asymptotically unbiased estimator over the previously proposed statistics: the mode and the mean. Additionally, from the probability density function, we derive the maximum likelihood formula for global intrinsic dimensionality, if i.i.d. holds. We tackle edge and finite-sample effects with an exponential correction formula, calibrated on hypercube datasets. We compare the performance of the corrected median-FSA estimator with kNN estimators: maximum likelihood (Levina-Bickel), the 2NN and two implementations of DANCo (R and MATLAB). We show that corrected median-FSA estimator beats the maximum likelihood estimator and it is on equal footing with DANCo for standard synthetic benchmarks according to mean percentage error and error rate metrics. With the median-FSA algorithm, we reveal diverse changes in the neural dynamics while resting state and during epileptic seizures. We identify brain areas with lower-dimensional dynamics that are possible causal sources and candidates for being seizure onset zones.

Single-Agent and Fixed-Dose Combination HIV-1 Protease Inhibitor Drugs in Fission Yeast (Schizosaccharomyces pombe).

Successful combination antiretroviral therapies (cART) eliminate active replicating HIV-1, slow down disease progression, and prolong lives. However, cART effectiveness could be compromised by the emergence of viral multidrug resistance, suggesting the need for new drug discoveries. The objective of this study was to further demonstrate the utility of the fission yeast cell-based systems that we developed previously for the discovery and testing of HIV protease (PR) inhibitors (PIs) against wild-type or multi-PI drug resistant M11PR that we isolated from an infected individual. All thirteen FDA-approved single-agent and fixed-dose combination HIV PI drugs were tested. The effect of these drugs on HIV PR activities was tested in pure compounds or formulation drugs. All FDA-approved PI drugs, except for a prodrug FPV, were able to suppress the wild-type PR-induced cellular and enzymatic activities. Relative drug potencies measured by EC50 in fission yeast were discussed in comparison with those measured in human cells. In contrast, none of the FDA-approved drugs suppressed the multi-PI drug resistant M11PR activities. Results of this study show that fission yeast is a reliable cell-based system for the discovery and testing of HIV PIs and further demonstrate the need for new PI drugs against viral multi-PI resistance.

Proteomic analysis of meiosis and characterization of novel short open reading frames in the fission yeast Schizosaccharomyces pombe.

Meiosis is the process by which haploid gametes are produced from diploid precursor cells. We used stable isotope labeling by amino acids in cell culture (SILAC) to characterize the meiotic proteome in the fission yeast Schizosaccharomyces pombe. We compared relative levels of proteins extracted from cells harvested around meiosis I with those of meiosis II, and proteins from premeiotic S phase with the interval between meiotic divisions, when S phase is absent. Our proteome datasets revealed peptides corresponding to short open reading frames (sORFs) that have been previously identified by ribosome profiling as new translated regions. We verified expression of selected sORFs by Western blotting and analyzed the phenotype of deletion mutants. Our data provide a resource for studying meiosis that may help understand differences between meiosis I and meiosis II and how S phase is suppressed between the two meiotic divisions.

A distinct class of plant and animal viral proteins that disrupt mitosis by directly interrupting the mitotic entry switch Wee1-Cdc25-Cdk1.

Many animal viral proteins, e.g., Vpr of HIV-1, disrupt host mitosis by directly interrupting the mitotic entry switch Wee1-Cdc25-Cdk1. However, it is unknown whether plant viruses may use this mechanism in their pathogenesis. Here, we report that the 17K protein, encoded by barley yellow dwarf viruses and related poleroviruses, delays G2/M transition and disrupts mitosis in both host (barley) and nonhost (fission yeast, Arabidopsis thaliana, and tobacco) cells through interrupting the function of Wee1-Cdc25-CDKA/Cdc2 via direct protein-protein interactions and alteration of CDKA/Cdc2 phosphorylation. When ectopically expressed, 17K disrupts the mitosis of cultured human cells, and HIV-1 Vpr inhibits plant cell growth. Furthermore, 17K and Vpr share similar secondary structural feature and common amino acid residues required for interacting with plant CDKA. Thus, our work reveals a distinct class of mitosis regulators that are conserved between plant and animal viruses and play active roles in viral pathogenesis.

Shed Light in the DaRk LineagES of the Fungal Tree of Life-STRES.

The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the "Shed light in The daRk lineagES of the fungal tree of life" (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments.

Development of A Fission Yeast Cell-Based Platform for High Throughput Screening of HIV-1 Protease Inhibitors.

BACKGROUND: HIV-1 protease inhibitor (PI) is one of the most potent classes of drugs in combinational antiretroviral therapies (cART). When a PI is used in combination with other anti- HIV drugs, cART can often suppress HIV-1 below detection thus prolonging the patient's lives. However, the challenge often faced by patients is the emergence of HIV-1 drug resistance. Thus, PIs with high genetic-barrier to drug-resistance are needed. OBJECTIVE: The objective of this study was to develop a novel and simple fission yeast (Schizosaccharomyces pombe) cell-based system that is suitable for high throughput screening (HTS) of small molecules against HIV-1 protease (PR). METHODS: A fission yeast RE294-GFP strain that stably expresses HIV-1 PR and green fluorescence protein (GFP) under the control of an inducible nmt1 promoter was used. Production of HIV-1 PR induces cellular growth arrest, which was used as the primary endpoint for the search of PIs and was quantified by an absorbance-based method. Levels of GFP production were used as a counter-screen control to eliminate potential transcriptional nmt1 inhibitors. RESULTS: Both the absorbance-based HIV-1 PR assay and the GFP-based fluorescence assay were miniaturized and optimized for HTS. A pilot study was performed using a small drug library mixed with known PI drugs and nmt1 inhibitors. With empirically adjusted and clearly defined double-selection criteria, we were able to correctly identify the PIs and to exclude all hidden nmt1 inhibitors. CONCLUSION: We have successfully developed and validated a fission yeast cell-based HTS platform for the future screening and testing of HIV-1 PR inhibitors.

Causal relationship between local field potential and intrinsic optical signal in epileptiform activity in vitro.

The directed causal relationship were examined between the local field potential (LFP) and the intrinsic optical signal (IOS) during induced epileptiform activity in in vitro cortical slices by the convergent cross-mapping causality analysis method. Two components of the IOS signal have been distinguished: a faster, activity dependent component (IOSh) which changes its sign between transmitted and reflected measurement, thus it is related to the reflectance or the scattering of the tissue and a slower component (IOSl), which is negative in both cases, thus it is resulted by the increase of the absorption of the tissue. We have found a strong, unidirectional, delayed causal effect from LFP to IOSh with 0.5-1s delay, without signs of feedback from the IOSh to the LFP, while the correlation was small and the peaks of the cross correlation function did not reflect the actual causal dependency. Based on these observations, a model has been set up to describe the dependency of the IOSh on the LFP power and IOSh was reconstructed, based on the LFP signal. This study demonstrates that causality analysis can lead to better understanding the physiological interactions, even in case of two data series with drastically different time scales.

Strategic Positioning of Connexin36 Gap Junctions Across Human Retinal Ganglion Cell Dendritic Arbors.

Connexin36 (Cx36) subunits form gap junctions (GJ) between neurons throughout the central nervous system. Such GJs of the mammalian retina serve the transmission, averaging and correlation of signals prior to conveying visual information to the brain. Retinal GJs have been exhaustively studied in various animal species, however, there is still a perplexing paucity of information regarding the presence and function of human retinal GJs. Particularly little is known about GJ formation of human retinal ganglion cells (hRGCs) due to the limited number of suitable experimental approaches. Compared to the neuronal coupling studies in animal models, where GJ permeable tracer injection is the gold standard method, the post-mortem nature of scarcely available human retinal samples leaves immunohistochemistry as a sole approach to obtain information on hRGC GJs. In this study Lucifer Yellow (LY) dye injections and Cx36 immunohistochemistry were performed in fixed short-post-mortem samples to stain hRGCs with complete dendritic arbors and locate dendritic Cx36 GJs. Subsequent neuronal reconstructions and morphometric analyses revealed that Cx36 plaques had a clear tendency to form clusters and particularly favored terminal dendritic segments.

Antagonistic interaction of HIV-1 Vpr with Hsf-mediated cellular heat shock response and Hsp16 in fission yeast (Schizosaccharomyces pombe).

BACKGROUND: Expression of the HIV-1 vpr gene in human and fission yeast cells displays multiple highly conserved activities, which include induction of cell cycle G2 arrest and cell death. We have previously characterized a yeast heat shock protein 16 (Hsp16) that suppresses the Vpr activities when it is overproduced in fission yeast. Similar suppressive effects were observed when the fission yeast hsp16 gene was overexpressed in human cells or in the context of viral infection. In this study, we further characterized molecular actions underlying the suppressive effect of Hsp16 on the Vpr activities. RESULTS: We show that the suppressive effect of Hsp16 on Vpr-dependent viral replication in proliferating T-lymphocytes is mediated through its C-terminal end. In addition, we show that Hsp16 inhibits viral infection in macrophages in a dose-dependent manner. Mechanistically, Hsp16 suppresses Vpr activities in a way that resembles the cellular heat shock response. In particular, Hsp16 activation is mediated by a heat shock factor (Hsf)-dependent mechanism. Interestingly, vpr gene expression elicits a moderate increase of endogenous Hsp16 but prevents its elevation when cells are grown under heat shock conditions that normally stimulate Hsp16 production. Similar responsive to Vpr elevation of Hsp and counteraction of this elevation by Vpr were also observed in our parallel mammalian studies. Since Hsf-mediated elevation of small Hsps occurs in all eukaryotes, this finding suggests that the anti-Vpr activity of Hsps is a conserved feature of these proteins. CONCLUSION: These data suggest that fission yeast could be used as a model to further delineate the potential dynamic and antagonistic interactions between HIV-1 Vpr and cellular heat shock responses involving Hsps.

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

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 combat mdrPRs. RESULTS: 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. CONCLUSIONS: 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.

Fission yeast homologue of Tip41-like proteins regulates type 2A phosphatases and responses to nitrogen sources.

A fission yeast (Schizosaccharomyces pombe) gene encoding a member of the TIP41-like protein family was identified and characterized. Deletion of the fission yeast tip41 gene leads to slower growth when ammonium chloride is the nitrogen source, but the growth rate is not affected when adenine is the nitrogen source. The tip41 mutant cells also enter the G1 phase of the cell cycle earlier than wild-type cells in response to nitrogen starvation. Overexpression of tip41(+) causes cell death, spherical cell morphology and blocks the shift to G1 phase upon nitrogen starvation. Overexpression of tip41(+) increases the activity of type 2A phosphatase. In a ppa2 deletion strain with reduced PP2A activity, overexpression of tip41(+) no longer blocks the shift to G1 upon nitrogen starvation. These results suggest that fission yeast Tip41 plays a role in cellular responses to nitrogen nutrient conditions at least partly through regulation of type 2A phosphatase activity.

HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe.

BACKGROUND: HIV-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 with cellular proteins and its potential impact on cell proliferation and viability. RESULTS: A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8. CONCLUSIONS: This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.