The genomes of positive-strand RNA [(؉)RNA] viruses perform two mutually exclusive functions: they act as mRNAs for the translation of viral proteins and as templates for viral replication. A universal key step in the replication of (؉)RNA viruses is the coordinated transition of the RNA genome from the cellular translation machinery to the viral replication complex. While host factors are involved in this step, their nature is largely unknown. By using the ability of the higher eukaryotic (؉)RNA virus brome mosaic virus (BMV) to replicate in yeast, we previously showed that the host Lsm1p protein is required for efficient recruitment of BMV RNA from translation to replication. Here we show that in addition to Lsm1p, all tested components of the Lsm1p-7p/Pat1p/Dhh1p decapping activator complex, which functions in deadenylation-dependent decapping of cellular mRNAs, are required for BMV RNA recruitment for RNA replication. In contrast, other proteins of the decapping machinery, such as Edc1p and Edc2p from the deadenylation-dependent decapping pathway and Upf1p, Upf2p, and Upf3p from the deadenylation-independent decapping pathway, had no significant effects. The dependence of BMV RNA recruitment on the Lsm1p-7p/Pat1p/Dhh1p complex was linked exclusively to the 3 noncoding region of the BMV RNA. Collectively, our results suggest that the Lsm1p-7p/Pat1p/Dhh1p complex that transfers cellular mRNAs from translation to degradation might act as a key regulator in the switch from BMV RNA translation to replication.Positive-strand RNA [(ϩ)RNA] viruses include important plant, animal, and human pathogens such as the severe acute respiratory syndrome coronavirus and hepatitis C virus. This large group of viruses replicate in the cytoplasm through negative-strand intermediates and share some fundamental features in their replication processes. A key common feature is the function of (ϩ)RNA virus genomes as templates for both translation and replication. In contrast to other virus groups, (ϩ)RNA viruses do not encapsidate viral polymerases required for viral replication, so upon virus entry into the cell, the genomic RNA must first be translated to produce viral replication factors. These replication factors then specifically recognize the viral RNA and recruit it from translation into the RNA replication complex. These two genomic RNA functions are mutually exclusive because 5Ј-to-3Ј ribosome trafficking blocks 3Ј-to-5Ј polymerase copying of viral (ϩ)RNA (5, 17). Therefore, the switch from genomic RNA translation to replication must be highly regulated to allow sufficient translation but also efficient replication. The molecular features underlying this regulation are poorly understood. Other important common features in the replication of (ϩ)RNA viruses are the assembly of replication complexes on intracellular membranes (31) and the requirement for host factors in multiple steps of the replication process (2). The identification of such host factors is important for a better understanding of fundamental issues in (ϩ)RNA ...

When DNA replication is challenged cells activate a DNA synthesis checkpoint, blocking cell cycle progression until they are able to overcome the replication defects. In fission yeast, Cds1 is the effector kinase of this checkpoint, inhibiting M‐phase entry, stabilizing stalled replication forks and triggering transcriptional activation of S‐phase genes. The molecular basis of this last effect is largely unknown. The Mlu1 binding factor (MBF) complex controls the transcription of S‐phase genes. We purified novel interactors of the MBF complex and identified the repressor Yox1. When the DNA synthesis checkpoint is activated, Yox1 is phosphorylated, which abrogates its binding to MBF. MBF‐dependent transcription therefore remains active until cells are able to overcome this challenge.

LSm1-7 complexes promote cellular mRNA degradation, in addition to translation and replication of positive-strand RNA viruses such as the Brome mosaic virus (BMV). Yet, how LSm1-7 complexes act on their targets remains elusive. Here, we report that reconstituted recombinant LSm1-7 complexes directly bind to two distinct RNA-target sequences in the BMV genome, a tRNA-like structure at the 39-untranslated region and two internal A-rich single-stranded regions. Importantly, in vivo analysis shows that these sequences regulate the translation and replication of the BMV genome. Furthermore, both RNA-target sequences resemble those found for Hfq, the LSm counterpart in bacteria, suggesting conservation through evolution. Our results provide the first evidence that LSm1-7 complexes interact directly with viral RNA genomes and open new perspectives in the understanding of LSm1-7 functions.

Understanding the fundamental steps of virus life cycles including virus-host interactions is essential for the design of effective antiviral strategies. Such understanding has been deferred by the complexity of higher eukaryotic host organisms. To circumvent experimental difficulties associated with this, systems were developed to replicate viruses in the yeast Saccharomyces cerevisiae. The systems include viruses with RNA and DNA genomes that infect plants, animals and humans. By using the powerful methodologies available for yeast genetic analysis, fundamental processes occurring during virus replication have been brought to light. Here, we review the different viruses able to direct replication and gene expression in yeast and discuss their main contributions in the understanding of virus biology.

DNA damage and DNA replication checkpoints regulate differently the G1-to-S phase transcriptional program, resulting in the repression or induction, respectively, of the same set of genes. When this signaling is disrupted, cells are unable to cope with DNA-damaging agents, leading to increased cell lethality.

The yeast Saccharomyces cerevisiae is a well-established model system for understanding fundamental cellular processes relevant to higher eukaryotic organisms. Less known is its value for virus research, an area in which Saccharomyces cerevisiae has proven to be very fruitful as well. The present review will discuss the main achievements of yeast-based studies in basic and applied virus research. These include the analysis of the function of individual proteins from important pathogenic viruses, the elucidation of key processes in viral replication through the development of systems that allow the replication of higher eukayotic viruses in yeast, and the use of yeast in antiviral drug development and vaccine production.

In the fission yeast Schizosaccharomyces pombe, meiosis is inhibited by the protein kinase Pat1, which phosphorylates and inactivates Mei2, an RNA binding protein essential for the initiation of meiosis. When diploid cells are deprived of nutrients, they initiate a cascade of events leading to the inactivation of Pat1 and entry into meiosis. Strains carrying the temperature-sensitive pat1-114 allele are forced to enter into meiosis when shifted to the non-permissive temperature, independently of the ploidity of the cell. This system has been extensively used, since it is possible to achieve a highly synchronous meiosis, which is a must for any molecular or microscopic approach that aims to decipher the mechanisms governing meiosis. Here, we have designed a new system to obtain a similarly synchronous meiosis, but independently of temperature shifts. Thus, by introducing a mutation in the ATP pocket of Pat1, we have generated a protein kinase that, in the presence of small specific inhibitors, can be inactivated. This results in forced entry into meiosis without the need of a temperature shift, minimizing the introduction of heat shock or any other stress responses along the meiotic waves of transcription.

By using a Brome mosaic virus (BMV)-Saccharomyces cerevisiae system, we previously showed that the cellular Lsm1p-7p/Pat1p/Dhh1p decapping-activator complex functions in BMV RNA translation and replication. As a first approach in investigating whether the corresponding human homologues play a similar role, we expressed human Lsm1p (hLsm1p) and RCK/p54 in yeast. Expression of RCK/p54 but not hLsm1p restored the defect in BMV RNA translation and replication observed in the dhh1⌬ and lsm1⌬ strains, respectively. This functional conservation, together with the common replication strategies of positive-stranded RNA viruses, suggests that RCK/p54 may also play a role in the replication of positive-stranded RNA viruses that infect humans.The group of positive-stranded RNA viruses includes major human pathogens such as hepatitis C virus and severe acute respiratory syndrome coronavirus. Since positive-stranded RNA viruses do not encapsidate the viral polymerase, upon entering the cell the viral RNA is first translated to produce viral replicases. Then, the viral RNA is recruited to membraneassociated replication complexes and used as a template for replication. This transition from translation to replication is a key step mediated by viral proteins as well as host factors (9, 15).The replication of Brome mosaic virus (BMV) in the yeast Saccharomyces cerevisiae is a well-established model system for studying common steps of positive-stranded RNA virus biology in a relatively simple genetic background (12). BMV is a plant virus with a tripartite positive-stranded RNA genome (1). RNA1 and RNA2 encode the replicases 1a, a helicase, and 2a, the polymerase. In the absence of 2a, 1a recruits the BMV positive-stranded RNA templates out of translation and into the endoplasmic reticulum, the site of replication. This recruitment dramatically increases the stability and accumulation of BMV positive-stranded RNAs (2,11,22). Finally, RNA3 encodes the movement and the capsid proteins. In this system, translation effects can easily be measured by following the translation efficiency of BMV RNA2 via Western blotting with 2a-specific antibodies (17), while recruitment of the viral RNA out of translation for replication can be studied via 1a-dependent RNA3 accumulation in Northern blots (12,15,19,20).With this BMV-yeast model system, we have recently shown that the yeast Lsm1p-7p/Pat1p/Dhh1p decapping-activator complex, which functions in cellular mRNA degradation (6), is required for both translation and recruitment of BMV RNA to the replication complex (8,15,17). The Lsm1p-7p heptameric ring and the helicase Dhh1p belong to the highly conserved families of Sm/Sm-like proteins and DEAD-box helicase, respectively (13, 23). All the components of the Lsm1p-7p/Pat1p/ Dhh1p complex localize in cytoplasmic foci called P bodies. These are dynamic structures and sites of mRNA degradation, mRNA storage, and translation control (3, 6, 7). Interestingly, the Lsm1p-7p/Pat1p/Dhh1p complex mediates the movement of cellular mRNAs out of translation in...