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Biochimica et Biophysica Acta 1783 (2008) 1413–1417 www.elsevier.com/locate/bbamcr

Review Viral induced ⁎ Manfred J. Schmitt , Jochen Reiter

Molecular and Biology, FR 8.3 Biosciences, Building A 1.5, Saarland University, D-66041 Saarbrücken, Germany Received 4 October 2007; received in revised form 17 January 2008; accepted 18 January 2008 Available online 7 February 2008

Abstract

In an analogous system to mammals, induction of an apoptotic cell death programme (PCD) in yeast is not only restricted to various exogenous factors and stimuli, but can also be triggered by viral killer toxins and viral pathogens. In yeast, toxin secreting killer strains are frequently infected with double-stranded (ds)RNA that are responsible for killer phenotype expression and toxin in the infected host. In most cases, the viral toxins are either pore-forming proteins (such as K1, K2, and zygocin) that kill non-infected and sensitive yeast cells by disrupting cytoplasmic membrane function, or protein toxins (such as K28) that act in the nucleus by blocking DNA synthesis and subsequently causing a G1/S cell cycle arrest. Interestingly, while all these toxins cause necrotic cell death at high concentration, they trigger caspase- and ROS-mediated apoptosis at low-to-moderate concentration, indicating that even low toxin doses are deadly by triggering PCD in enemy cells. Remarkably, viral toxins are not solely responsible for cell death induction in vivo, as killer viruses themselves were shown to trigger apoptosis in non-infected yeast. Thus, as killer virus-infected and toxin secreting are effectively protected and immune to their own toxin, killer yeasts bear the intrinsic potential to dominate over time in their natural habitat. © 2008 Elsevier B.V. All rights reserved.

Keywords: Yeast; Apoptosis; Killer toxin; dsRNA virus

1. Introduction [2,4,24,29]. In the yeast , three different killer toxins have been identified so far (K1, K2, K28), all of In higher eukaryotes and mammals, virus infections are known which are genetically encoded by cytoplasmic persisting double- to induce a programmed cell death (PCD) pathway as first line of stranded RNA viruses encoding an unprocessed precursor of a defense in preventing virus transmission. Most remarkably, secreted α/β protein toxin [34,40]. Most viral killer toxins, like the unicellular organisms such as yeast likewise respond to viral S. cerevisiae K1 toxin and the Zygosaccharomyces bailii toxin pathogens and virus toxins, indicating that PCD is evolutionarily zygocin, act as ionophores and disrupt cytoplasmic membrane conserved and most likely arose before multicellular organisms function by forming cation-specific plasma membrane pores have developed [15,27]. Interestingly, the onset of yeast apoptosis [25,37,38]. In contrast however, the S. cerevisiae K28 toxin enters was recently shown to also have an effect on the assembly- susceptible cells by -mediated endocytosis, travels the dependent maturation of insect tetraviral procapsids in vivo [35]. secretion pathway in reverse, and finally acts in the nucleus by In yeast, the production of virally encoded, cytotoxic proteins irreversibly blocking DNA synthesis and causing a G1/S cell (so-called killer toxins) is a widespread phenomenon and typically cycle arrest [7,13,31]. associated with the secretion of protein toxins that kill susceptible In higher multicellular organisms it is well known that pore- target (yeast) cells in a two-step receptor-mediated fashion forming toxins like Staphylococcus aureus alpha toxin and/or inhibitors of protein synthesis like diphtheria toxin produced and secreted by Corynebacterium diphtheriae induce apoptosis [39]. The initial finding of cell death with apoptosis-like fea- ⁎ Corresponding author. Molecular and Cell Biology (FR 8.3 Biosciences), Saarland University, PO Box 15 11 50, Campus Building A 1.5, D-66041 tures in unicellular yeast [21] was unexpected and it was Saarbrücken, Germany. Tel.: +49 681 302 4730; fax: +49 681 302 4710. doubted that single-cell organisms would have any advantage in E-mail address: [email protected] (M.J. Schmitt). committing suicide. However, intensive studies of apoptotic

0167-4889/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2008.01.017 1414 M.J. Schmitt, J. Reiter / Biochimica et Biophysica Acta 1783 (2008) 1413–1417 phenomena in S. cerevisiae demonstrated that programmed cell 2. Killer yeasts and viral killer toxins death in yeast can be induced by a great number of exogenous and intrinsic stresses such as H2O2, UV-irradiation, acetic dsRNA viruses in yeast were initially discovered as deter- acid, cell aging, high pheromone concentration, defects in pro- minants of the killer phenomenon and are now known to be tein N-, antimicrobial peptides, and many more associated with the presence of cytoplasmically inherited my- scenarios [6,12,14,17,20,22,26,32]. In the majority of cases coviruses, which are frequently found in different yeast genera. describing an apoptotic phenotype in yeast it was shown that Among these, killers of S. cerevisiae, Z. bailii, Hanseniaspora debilitated cells die for the benefit of the whole yeast population uvarum and Ustilago maydis – the latter being the cause of corn saving limited nutrients for healthy cells in order to enable smut – have been most intensively studied. Characteristic for survival of the whole population [9,14]. Similar to mammalian all killers is the secretion of a particular protein toxin that is lethal apoptosis, reactive oxygen species (ROS) and mitochondria are to sensitive strains of different species and genera. Cell killing is central to most of these scenarios, and the striking similarity usually achieved in a receptor-mediated process, requiring initial between yeast and mammalian apoptosis was further underlined toxin binding to receptors within the outer yeast cell surface by the finding of yeast orthologues of mammalian pro- and anti- (such as β-1,6-D-glucan, α-1,3-mannoprotein or chitin) and apoptotic factors such as the metacaspase Yca1p [23], the subsequent transfer to a plasma membrane receptor. Depending HtrA2/Omi homologue Nma111p [8], an inhibitor-of-apoptosis on the type of toxin, lethality can either be caused by plasma (IAP) [36], the transkingdom Bax inhibitor-1 protein Bir1p [5] membrane damage, G1/S cell cycle arrest and/or rapid inhibition and two AIF/AMID homologues, the pro-apoptotic serine of DNA synthesis [30] (Fig. 1). protease Aif1p and Ndi1p [19,41]. However, despite all these In S. cerevisiae, Z. bailii and H. uvarum as well as in the maize proteins were shown to function in yeast apoptosis under certain smut fungus U. maydis, the killer phenotype is cytoplasmically conditions, natural pathways in which these proteins are es- inherited and caused by an infection with dsRNA viruses of the sentially involved have not yet been identified, and target(s) of family Totiviridae that are widely distributed among yeasts and yeast metacaspase Yca1p still remain to be elucidated [11]. higher fungi. Since the majority of fungal viruses are non-

Fig. 1. Receptor-mediated toxicity of yeast viral killer toxins K28, K1 and zygocin. Killing of a sensitive target (yeast) cell by each virus toxin is envisaged in a two- step process involving initial toxin binding to receptors within the cell wall (R1) and the cytoplasmic membrane (R2). After interaction with the plasma membrane, ionophoric toxins such as K1 and zygocin disrupt plasma membrane integrity, while K28 enters the cell by endocytosis and diffuses into the nucleus to cause non-PCD [note that the cell surface receptors R1 and R2 are different in all three toxins; see also table inset]. At high dose of K28 toxin (N10 pM), sensitive cells arrest in the cell cycle with pre-replicated DNA (1n; left panel), while low concentrations of K28 (b1 pM) activate a programmed cell death pathway exhibiting typical apoptotic markers such as chromosomal DNA fragmentation (TUNEL positive cells), accumulation of reactive oxygen species (ROS) and phosphatidylserine exposure at the external surface of the plasma membrane detected by annexin V staining (mid panel). Host cell proteins involved in toxin-mediated PCD (Kre1p, Tok1p, Yca1p, Dnm1p) or non-PCD (Kre1p) are indicated (the model is based on data from Reiter et al. [27] and Ivanovska and Hardwick [15]). M.J. Schmitt, J. Reiter / Biochimica et Biophysica Acta 1783 (2008) 1413–1417 1415 infectious and symptomless in the corresponding host, they are typically accompanied by DNA fragmentation, chromatin con- often classified as cryptic viruses. All known fungal viruses densation and phosphatidylserine externalization (Fig. 1). This spread vertically by cell-to-cell mating and/or heterokaryon for- process is mediated through yeast caspase Yca1p and the mation. In S. cerevisiae, diploids formed by mating of a killer with generation of ROS [27]. In contrast, high toxin concentrations a sensitive strain are likewise killers, as are all haploid progeny (N10 pM) induce non-apoptotic (necrotic) cell death indepen- of subsequent meiosis. In contrast, non-infected and virus-free dent of Yca1p and ROS. Therefore, killer toxin action can trigger strains are usually sensitive non-killers, while those containing a two modes of cell death: at high concentration, toxin-induced toxin-encoding killer virus and a helper virus (the latter providing apoptosis plays a minor role, while low-to-moderate toxin doses both viruses with Gag and Gag/Pol for viral RNA replication and trigger PCD in susceptible target cells. Since low toxin concen- packaging) survive mating with killers, and cytoplasmic mixing trations resemble the in vivo situation in the natural habitat, of the multiple killer viruses during zygosis accounts for the toxin-induced apoptotic host cell responses seem to be inheritance pattern during meiosis. Extracellular virus spread in particularly important at toxin concentrations that per se are yeast is largely hampered by the rigid cell wall barrier and fungal too low to kill via the toxin's primary mode of action. viruses, therefore, adopted a strategy of transmission via mating In contrast to rather “artificial” stresses inducing PCD in yeast and hyphal fusion which frequently occurs in nature, making an (such as high pheromone concentrations), a more “natural” cell extracellular route of spread dispensible. Concomitant with killer death scenario resembles the exposure of yeast to cytotoxic phenotype expression, toxin production is usually associated with proteins produced and secreted by concurring killer strains. specific immunity effectively protecting killer yeasts against their Under these conditions – which frequently occur in the natural own toxin [3]. environment of yeast and higher fungi – it was recently de- monstrated that viral killer toxins induce an apoptotic cell death 3. Apoptosis as natural part in yeast killer toxin lethality pathway in sensitive cells, in particular under conditions that closely reflect the in vivo situation in the natural yeast habitat In the majority of yeast killer toxins, the primary mode of [27]. Most interestingly and completely independent of the killing either involves disruption of plasma membrane integrity actual molecular mode of cell killing, all viral killer toxins were and function, or interference with essential host cell proteins that shown to induce apoptotic cell death at low-to-moderate con- are involved in eukaryotic cell cycle control and/or chromoso- centration (b1 pM). Thus, no matter whether a virus toxin mal DNA synthesis. Such a scenario particularly occurs under disrupts plasma membrane function or causes a G1/S cell cycle high toxin conditions (see below) under which sensitive cells are arrest, it always induces apoptotic phenotypes that are accom- either killed within a few minutes after toxin exposure (as is the panied by the production of reactive oxygen species (ROS), case with ionophoric killer toxins [38]) or within the frame of a chromosomal DNA fragmentation, typical phenotypic changes, single cell cycle; the latter mode of toxicity is characteristic for and translocation of phosphatidylserine from the inner to the the K28 virus toxin and the -encoded killer toxin of outer leaflet of the cytoplasmic membrane, the defining Kluyveromyces lactis [18,28]. However, at low toxin concentra- phenotype in PCD [27]. Correspondingly, Δyca1 null mutant tions (reflecting the natural environmental situation [33]), viral cells which lack the only metacaspase in yeast (Yca1p) are killer toxins induce an apoptotic cell death response that is significantly less sensitive to viral toxins, while mutants blocked

Fig. 2. Yeast apoptosis signaling triggered in non-infected sensitive cells by a K1 toxin secreting and killer virus-infected yeast. The indicated K1 killer harbors cytoplasmic persisting dsRNA viruses (helper and killer viruses) ensuring both, K1 toxin secretion and protecting K1 immunity. Within a non-infected target cell, low K1 toxin concentrations (b1 pM) and possibly additional factors secreted by the killer cell induce a PCD pathway whose intracellular signaling involves host proteins such as Tok1p, Kre1p, Yca1p and Dnm1p, finally leading to mitochondrial ROS production and apoptotic cell death. In contrast, PCD triggered by K1 and/or K1 encoding killer viruses is inhibited by the pore-forming mitochondrial outer membrane protein Fis1p [2,15,27]. 1416 M.J. Schmitt, J. Reiter / Biochimica et Biophysica Acta 1783 (2008) 1413–1417 in endogenous glutathione biosynthesis (such as Δgsh1) behave markers of mammalian apoptosis, might be the first example of hypersensitive. In contrast to the situation at low toxin concen- a natural occurring phenomenon that explains why even uni- tration, high toxin doses (N10 pM) cause non-apoptotic cell cellular organisms could benefit from a suicidal cell death death independent of either yeast caspase 1 or ROS. Interest- programme. Killer toxin production and secretion resembles a ingly, very similar effects were likewise shown in yeast after special kind of yeast “warfare” in which killer yeasts activate a treatment with a plasmid-driven killer toxin secreted by the yeast self-destruct mechanism in virus-free and non-self enemy cells. Pichia acaciae [16]. However, further research is needed to elucidate these and possibly additional pathways that regulate yeast apoptosis in 4. Yeast killer viruses induce apoptosis in non-infected response to microbial and viral protein toxins, dsRNA killer yeast viruses and other environmental stimuli, which somehow do have in common that they all regulate yeast cell populations. 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