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

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

Advanced Protocol for Molecular Characterization of Viral Genome in Fission Yeast (<i>Schizosaccharomyces pombe</i>)

Fission yeast, a single-cell eukaryotic organism, shares many fundamental cellular processes with higher eukaryotes, including gene transcription and regulation, cell cycle regulation, vesicular transport and membrane trafficking, and cell death resulting from the cellular stress response. As a result, fission yeast has proven to be a versatile model organism for studying human physiology and diseases such as cell cycle dysregulation and cancer, as well as autophagy and neurodegenerative diseases like Alzheimer’s, Parkinson’s, and Huntington’s diseases. Given that viruses are obligate intracellular parasites that rely on host cellular machinery to replicate and produce, fission yeast could serve as a surrogate to identify viral proteins that affect host cellular processes. This approach could facilitate the study of virus–host interactions and help identify potential viral targets for antiviral therapy. Using fission yeast for functional characterization of viral genomes offers several advantages, including a well-characterized and haploid genome, robustness, cost-effectiveness, ease of maintenance, and rapid doubling time. Therefore, fission yeast emerges as a valuable surrogate system for rapid and comprehensive functional characterization of viral proteins, aiding in the identification of therapeutic antiviral targets or viral proteins that impact highly conserved host cellular functions with significant virologic implications. Importantly, this approach has a proven track record of success in studying various human and plant viruses. In this protocol, we present a streamlined and scalable molecular cloning strategy tailored for genome-wide and comprehensive functional characterization of viral proteins in fission yeast

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

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

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 ( 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\u2193CA 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 ..

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

Contains fulltext : 228731.pdf (publisher's version ) (Open Access

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

Hhp2 suppresses <i>PR</i>-induced cell death and apoptosis in HeLa cells.

<p><b>(A)</b> Expression of HIV-1 <i>PR</i> induces apoptosis in HeLa cells. Apoptosis were detected by staining with Annexin V and FACScan analysis as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151286#pone.0151286.ref064" target="_blank">64</a>]. The percentage of apoptotic cells was measured in HeLa cells 24 hrs after transfection of a control (Ctr) pIRES2-EGFP plasmid (left) or a <i>PR</i>-containing pIRES2-EGFP-<i>PR</i> plasmid (right). The average number ± SD of apoptotic cells are shown in the top right quadrant (<b>a</b>), using data from at least three independent measurements. (<b>b</b>) Expression of HIV-1 PR induced caspase-3 and caspase-8 cleavage in HeLa cells as shown by western blot analyses performed as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151286#pone.0151286.ref025" target="_blank">25</a>]. Lane 1, HeLa cells transfected with a control plasmid; lane 2, <i>PR</i>-expressing transfected HeLa cells. The analysis was carried out 48 hrs after plasmid transfection. (<b>B</b>) Hhp2 suppresses <i>PR</i>-induced cell death and apoptosis in HeLa cells. Since no specific antibody against Hhp2 was available, to confirm <i>hhp2</i> gene expression in HeLa cells, <i>hhp2</i> gene transcription was measured by RT-PCR and detected on agarose gel as shown in (<b>a</b>). Lane 1, HeLa cells transfected with a control plasmid; lane 2, <i>hhp2</i>-expressing HeLa cells. The gel shows the 1.2-kb <i>hhp2</i> cDNA that was amplified. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The suppressive effect of Hhp2 on HIV-1 PR-induced cell death and apoptosis was also quantified 24 hrs after gene expression using a Trypan blue assay (<b>b</b>) and Annexin V staining (<b>c</b>), respectively. The average numbers ± SD of dead cells are shown, the error bars represent results of at least three independent measurements. The effect of Hhp2 on HIV-1 PR-mediated caspase-3 and caspase-8 cleavage was detected by western blot analysis as shown in (<b>d</b>) and quantified in (<b>e</b>). A statistical t-test was used to determine whether there was a significant difference in the cleavage of caspase-3 and caspase-8 taking place in the control HeLa cells and HIV-1 <i>PR</i>-expressing cells, with or without Hhp2. p-values less than 0.01 were considered statistically significant.</p