The RNA homologous to the yeast transposable element Tyl is one of the more abundant poly(A)+ RNAs in many strains of the yeast Saccharomyces cerevsWae. The 5' and 3' ends of Tyl RNA have been determined from analysis of cDNA. The 5' end is 245 bases into the left 6 sequence measured from the left side of the Tyl element. The 6 sequence is a direct repeat of about 340 base pairs present at each end of the Tyl element. The Tyl transcription includes 93-97 bases of the left 6 sequence and continues through the entire internal portion of the element and through about 295 bases of the right 6 sequence before reaching the 3' end located 38-46 bases from the right side of the right 6 sequence. Because the 6 sequences present at each end of a single Tyl element have identical or very similar DNA sequences, these end points for Tyl RNA raise several questions about the expression of Tyl elements. First, what are the initiation and termination signals, because the Tyl transcript must read through a DNA sequence that is identical to the 3' end at about 50 bases from the 5' end? Second, why is the direction of transcription of the Tyl element opposite to that of genes that are overexpressed after the insertion of a Tyl element? Third, because the Tyl RNA itself has direct repeats of about 45 (9,10). The exact number of base pairs generated is specific for the transposable element. For example, 5 bp are always generated by the yeast element Tyl and the Drosophila element copia (4, 5, 7). Another common characteristic of these elements is that they are heavily transcribed to give abundant poly(A)+ RNAs (2, 11).In addition to the abundant transcription of the element itself, Tyl can affect the expression of a gene flanking the element. Insertions of Tyl that inactivate the his4 gene have been studied thoroughly by [12][13][14]. Another class of mutations produces 3-50 times more of the gene product after the insertion of a Tyl element near the 5' end of the gene. Such Tyl insertion mutations that increase expression have been isolated for CYC7(15-17), ADR2 (18), HIS3 (19), CAR1 (20), DUR1 (21), and probably CAR2 (16). The insertion of aThe publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Tyl element is then able to increase the expression of the flanking gene for many different genes.Tyl RNA and the overexpression of a gene flanking a Tyl element show an unexpected relationship to the mating type of the yeast cell. In stationary a or a mating type cells, 5-10% of the poly(A)+ RNA is Tyl RNA, but in a/a cells, the amount of Tyl RNA is 95% lower (11). Tyl transposition mutations that overexpress the flanking gene show a similar dependence on the mating type of the cell because the amount of gene expression is reduced 75-95% in a/a cells compared to that in a or a cells (16). The Tyl element then contains the necessary signals to place both its own expression and t...

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The SP013 gene, required for meiosis I segregation in Saccharomyces cerevisiae, produces two developmentally regulated transcripts (1.0 and 1.4 kilobases) that differ in length at their 5' ends. The shorter transcript is sufficient to complement the spol3-1 mutation and contains a major open reading frame encoding a highly basic protein of 33.4 kilodaltons. A fragment upstream (-170 to -8) of the open reading frame confers meiosis-specific transcription on a spol3-HIS3 fusion. Deletions at the 5' end of spo3-lacZ fusions define a region between -140 and -80 that is essential for meiosis-specific expression. This region acts in an orientation-independent manner and is responsive to the MAT-RME regulatory cascade. It contains a 10-base-pair sequence, TAGCCGCCGA, found in a number of meiosis-specific genes, that appears to be required for SP013 expression. This sequence is identical to URS1, a ubiquitous mitotic repressor element.Initiation of meiosis and spore formation in Saccharomyces cerevisiae is controlled by cell type and nutritional status (1, 2). Cell-type control occurs through the products of the MATa and MATa loci, which combine to negatively regulate transcription of RMEI (3, 4). RMEI negatively regulates IMEI, whose expression appears to be sufficient to induce meiosis (5). IMEI is also under negative control by the system monitoring nutritional status (5, 6).SP013 is required for the proper separation of homologs at meiosis I (7). SP013 has been cloned and encodes two overlapping transcripts with the same 3' end (8 MATERIALS AND METHODS Strains. RE944 (spol3-1/spol3-1 ura3/ura3) is from our strain collection. LP112 (MATa/MATa ade2/ade2 canl-100/ canl-100 his3-1 1, 15/his3-1 1,15 leu2-3,1 12/leu2-3, 112 trpl-1/ trpl-l ura3-1/ura3-1), obtained from S. Lindquist (University of Chicago), is a cross of W303-1A and W303-1B from R. Rothstein (Columbia University). Isogenic diploids SFY67 (MATa/MATa) and SFY68 (MATa/MATa) were obtained from J. Segall (University of Toronto). S104 (MATa/MATa leul/LEUI ura3/ura3 trpl/trpl cani/cani his4-519/HIS4 RMEI/rmel::LEU2), S150 (MATa/MATa leu2/leu2 ura3/ ura3 trpl/trpl his4-519/his4-712 canl/CANl lysliL YSI RMEl/rmel::LEU2), S151 (MATa/MATa leu2/leu2 ura3/ ura3 trpl/trpl his4-519/his4-712 canl/CANI lysl/L YSI rmel ::LEU2/rmel ::LEU2), S152 (matal-50/MATa leu2/ leu2 ura3/ura3 trpl/trpl his4-519/his4-712 canl/CANI lysl/ LYSI RMEJ/rmel ::LEU2), and S153 (matal-50/MATa leu2/leu2 ura3/ura3 trpl/trpl his4-519/his4-712 canl/CANI lysl/LYSI rmel::LEU2/rmel::LEU2) were acquired from A. Mitchell (Columbia University).Growth and Sporulation. General methods for growth and sporulation have been described (7,8,14). Strains were grown to midlogarithmic phase in synthetic acetate medium [1% potassium acetate plus 0.68% Difco yeast nitrogen base without amino acids, buffered to pH 6.5 with 0.05 M potassium phthalate (pH 5.5) and supplemented with auxotrophic requirements] prior to sporulation.DNA Sequence Analysis. Dideoxy sequencing of the cloned SP013 gene was carried out as ...

Expression of HIV-1 Vpr causes cell cycle G2 arrest, change in cell shape, and cell death over a large evolutionary distance ranging from human to yeast cells. As a step toward understanding these highly conserved Vpr functions, we have examined the effect of Vpr on cytoskeletal elements and the viability of fission yeast. We demonstrate that the changes in cell morphology induced by Vpr in fission yeast are caused by several underlying cellular abnormalities, including increased biosynthesis of chitin in the cell wall, disruption of the actin cytoskeleton, and altered polarity for cell growth. The extent of these cellular alterations and cell survival correlates with the level of vpr expression. Accompanying cell death, Vpr induces aberrant nuclear morphologies in fission yeast which are similar to those found during the apoptosis induced by Vpr in mammalian cells. The Vpr-induced cytopathic effects and cell death can be suppressed by treatment with pentoxifylline, a compound that inhibits HIV-1 viral replication and suppresses Vpr-induced cell cycle G2 arrest in human and fission yeast cells. The results presented here suggest that pentoxifylline suppresses the effects of Vpr by blocking interactions of Vpr with cellular proteins. Given that pentoxifylline has potential therapeutic value in blocking the effects of Vpr in HIV-infected patients, understanding the molecular mechanisms by which pentoxifylline antagonizes Vpr may have general implications for HIV therapy.

Viral protein R (Vpr) of human immunodeficiency virus type 1 induces G2 arrest in cells from distantly related eukaryotes including human and fission yeast through inhibitory phosphorylation of tyrosine 15 (Tyr15) on Cdc2. Since the DNA damage and DNA replication checkpoints also induce G2 arrest through phosphorylation of Tyr15, it seemed possible that Vpr induces G2 arrest through the checkpoint pathways. However, Vpr does not use either the early or the late checkpoint genes that are required for G2 arrest in response to DNA damage or inhibition of DNA synthesis indicating that Vpr induces G2 arrest by an alternative pathway. It was found that protein phosphatase 2A (PP2A) plays an important role in the induction of G2 arrest by Vpr since mutations in genes coding for a regulatory or catalytic subunit of PP2A reduce Vpr-induced G2 arrest. Vpr was also found to upregulate PP2A, supporting a model in which Vpr activates the PP2A holoenzyme to induce G2 arrest. PP2A is known to interact genetically in fission yeast with the Wee1 kinase and Cdc25 phosphatase that act on Tyr15 of Cdc2. Both Wee1 and Cdc25 play a role in Vpr-induced G2 arrest since a wee1 deletion reduces Vpr-induced G2 arrest and a direct in vivo assay shows that Vpr inhibits Cdc25. Additional support for both Wee1 and Cdc25 playing a role in Vpr-induced G2 arrest comes from a genetic screen, which identified genes whose overexpression affects Vpr-induced G2 arrest. For this genetic screen, a strain was constructed in which cell killing by Vpr was nearly eliminated while the effect of Vpr on the cell cycle was clearly indicated by an increase in cell length. Overexpression of the wos2 gene, an inhibitor of Wee1, suppresses Vpr-induced G2 arrest while overexpression of rad25, an inhibitor of Cdc25, enhances Vpr-induced G2 arrest. These two genes may be part of the uncharacterized pathway for Vpr-induced G2 arrest in which Vpr upregulates PP2A to activate Wee1 and inhibit Cdc25.

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) exerts multiple effects on viral and host cellular activities during viral infection, including nuclear transport of the proviral integration complex, induction of cell cycle G 2 arrest, and cell death. In this report, we show that a fission yeast chaperone protein Hsp16 inhibits HIV-1 by suppressing these Vpr activities. This protein was identified through three independent genome-wide screens for multicopy suppressors of each of the three Vpr activities. Consistent with the properties of a heat shock protein, heat shock-induced elevation or overproduction of Hsp16 suppressed Vpr activities through direct protein-protein interaction. Even though Hsp16 shows a stronger suppressive effect on Vpr in fission yeast than in mammalian cells, similar effects were also observed in human cells when fission yeast hsp16 was expressed either in vpr-expressing cells or during HIV-1 infection, indicating a possible highly conserved Vpr suppressing activity. Furthermore, stable expression of hsp16 prior to HIV-1 infection inhibits viral replication in a Vpr-dependent manner. Together, these data suggest that Hsp16 inhibits HIV-1 by suppressing Vpr-specific activities. This finding could potentially provide a new approach to studying the contribution of Vpr to viral pathogenesis and to reducing Vpr-mediated detrimental effects in HIV-infected patients.

Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast (Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15 (Tyr15) on Cdc2, which is phosphorylated by Wee1 kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two wellcharacterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins, which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-1) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest. Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.

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 with cellular proteins and its potential impact on cell proliferation and viability.ResultsA 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.ConclusionsThis 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.