Rosa Esteban

A New Wine Saccharomyces cerevisiae Killer Toxin (Klus), Encoded by a Double-Stranded RNA Virus, with Broad Antifungal Activity Is Evolutionarily Related to a Chromosomal Host Gene

ABSTRACT Wine Saccharomyces cerevisiae strains producing a new killer toxin (Klus) were isolated. They killed all the previously known S. cerevisiae killer strains, in addition to other yeast species, including Kluyveromyces lactis and Candida albicans. The Klus phenotype is conferred by a medium-size double-stranded RNA (dsRNA) virus, Saccharomyces cerevisiae virus Mlus (ScV-Mlus), whose genome size ranged from 2.1 to 2.3 kb. ScV-Mlus depends on ScV-L-A for stable maintenance and replication. We cloned and sequenced Mlus. Its genome structure is similar to that of M1, M2, or M28 dsRNA, with a 5′-terminal coding region followed by two internal A-rich sequences and a 3′-terminal region without coding capacity. Mlus positive strands carry cis-acting signals at their 5′ and 3′ termini for transcription and replication similar to those of killer viruses. The open reading frame (ORF) at the 5′ portion codes for a putative preprotoxin with an N-terminal secretion signal, potential Kex2p/Kexlp processing sites, and N-glycosylation sites. No sequence homology was found either between the Mlus dsRNA and M1, M2, or M28 dsRNA or between Klus and the K1, K2, or K28 toxin. The Klus amino acid sequence, however, showed a significant degree of conservation with that of the product of the host chromosomally encoded ORF YFR020W of unknown function, thus suggesting an evolutionary relationship.

Yeast Positive-stranded Virus-like RNA Replicons 20 S AND 23 S RNA TERMINAL NUCLEOTIDE SEQUENCES AND 3′ END SECONDARY STRUCTURES RESEMBLE THOSE OF RNA COLIPHAGES

Saccharomyces cerevisiae strains carry single-stranded RNAs called 20 S RNA and 23 S RNA. These RNAs and their double-stranded counterparts, W and T dsRNAs, have been cloned and sequenced. A few nucleotides at both ends, however, remained unknown. These RNAs do not encode coat proteins but their own RNA-dependent RNA polymerases that share a high degree of conservation to each other. The polymerases are also similar to the replicases of RNA coliphages, such as Qβ. Here we have determined the nucleotide sequences of W and T dsRNAs at both ends using reverse transcriptase polymerase chain reaction-generated cDNA clones. We confirmed the terminal sequences by primer-extension and RNase protection experiments. Furthermore, these analyses demonstrated that W and T dsRNAs and their single-stranded RNA counterparts (i) are linear molecules, (ii) have identical nucleotide sequences at their ends, and (iii) have no poly(A) tails at their 3′ ends. Both 20 S and 23 S RNAs have GGGGC at the 5′ ends and the complementary 5-nucleotides sequence, GCCCC-OH, at their 3′ ends. S1 and V1 secondary structure-mapping of the 3′ ends of 20 S and 23 S RNAs shows the presence of a stem-loop structure that partially overlaps with the conserved 3′ end sequence. Nucleotide sequences and stem-loop structures similar to those described here have been found at the 3′ ends of RNA coliphages. These data, together with the similarity of the RNA-dependent RNA polymerases encoded among these RNAs and RNA coliphages, suggest that 20 S and 23 S RNAs are plus-strand single-stranded virus-like RNA replicons in yeast.

Genomic Organization of T and W, a New Family of Double-Stranded RNAs from Saccharomyces cerevisiae

Publisher Summary This chapter describes the characterization of two dsRNAs. Most of the information comes from cloning and sequencing of their cDNAs. Their nucleotide sequences reveal that they are of viral origin, but their non-encapsidated nature distinguishes them from other dsRNA viruses. T and W were first described as dsRNAs present in a number of different laboratory strains of S. cerevisiae; the sizes of T and W were estimated to be 2.7 and 2.25 kb, respectively, in agarose gels. Both T and W are cytoplasmically inherited and are not encapsidated into viral particles. Both are inducible in certain yeast strains when grown at 37°C. W is present in most laboratory yeast strains. The fact that T has always been found in W-carrying strains may suggest that T is dependent on W for its maintenance. Therefore, T and W dsRNAs are different from the other dsRNAs present in S. cerevisiae and they are maintained autonomously in the yeast cells, probably by an RNA-to-RNA replication pathway.

Launching of the yeast 20 s RNA narnavirus by expressing the genomic or antigenomic viral RNA in vivo

20 S RNA virus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome encodes only its RNA polymerase, p91, and resides in the cytoplasm in the form of a ribonucleoprotein complex with p91. We succeeded in generating 20 S RNA virus in vivo by expressing, from a vector, genomic strands fused at the 3′-ends to the hepatitis delta virus antigenomic ribozyme. Using this launching system, we analyzed 3′-cis-signals present in the genomic strand for replication. The viral genome has five-nucleotide inverted repeats at both termini (5′-GGGGC... GCCCC-OH). The fifth G from the 3′-end was dispensable for replication, whereas the third and fourth Cs were essential. The 3′-terminal and penultimate Cs could be eliminated or modified to other nucleotides; however, the generated viruses recovered these terminal Cs. Furthermore, extra nucleotides added at the viral 3′-end were eliminated in the launched viruses. Therefore, 20 S RNA virus has a mechanism(s) to maintain the correct size and sequence of the viral 3′-end. This may contribute to its persistent infection in yeast. We also succeeded in generating 20 S RNA virus similarly from antigenomic strands provided active p91 was supplied from a second vector in trans. Again, a cluster of four Cs at the 3′-end in the antigenomic strand was essential for replication. In this work, we also present the first conclusive evidence that 20 S and 23 S RNA viruses are independent replicons.

Native Replication Intermediates of the Yeast 20 S RNA Virus Have a Single-stranded RNA Backbone

20 S RNA virus is a positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome (2.5 kb) only encodes its RNA polymerase (p91) and forms a ribonucleoprotein complex with p91 in vivo. A lysate prepared from 20 S RNA-induced cells showed an RNA polymerase activity that synthesized the positive strands of viral genome. When in vitro products, after phenol extraction, were analyzed in a time course, radioactive nucleotides were first incorporated into double-stranded RNA (dsRNA) intermediates and then chased out to the final single-stranded RNA products. The positive and negative strands in these dsRNA intermediates were non-covalently associated, and the release of the positive strand products from the intermediates required a net RNA synthesis. We found, however, that these dsRNA intermediates were an artifact caused by phenol extraction. Native replication intermediates had a single-stranded RNA backbone as judged by RNase sensitivity experiments, and they migrated distinctly from a dsRNA form in non-denaturing gels. Upon completion of RNA synthesis, positive strand RNA products as well as negative strand templates were released from replication intermediates. These results indicate that the native replication intermediates consist of a positive strand of less than unit length and a negative strand template loosely associated, probably through the RNA polymerase p91. Therefore, W, a dsRNA form of 20 S RNA that accumulates in yeast cells grown at 37 °C, is not an intermediate in the 20 S RNA replication cycle, but a by-product.

Recognition of RNA Encapsidation Signal by the Yeast L-A Double-stranded RNA Virus

The encapsidation signal of the yeast L-A virus contains a 24-nucleotide stem-loop structure with a 5-nucleotide loop and an A bulged at the 5′ side of the stem. The Pol part of the Gag-Pol fusion protein is responsible for encapsidation of viral RNA. Opened empty viral particles containing Gag-Pol specifically bind to this encapsidation signal in vitro. We found that binding to empty particles protected the bulged A and the flanking-two nucleotides from cleavage by Fe(II)-EDTA-generated hydroxyl radicals. The five nucleotides of the loop sequence (4190GAUCC4194) were not protected. However, T1 RNase protection and in vitro mutagenesis experiments indicated that G4190 is essential for binding. Although the sequence of the other four nucleotides of the loop is not essential, data from RNase protection and chemical modification experiments suggested that C4194 was also directly involved in binding to empty particles rather than indirectly through its potential base pairing with G4190. These results suggest that the Pol domain of Gag-Pol contacts the encapsidation signal at two sites: one, the bulged A, and the other, G and C bases at the opening of the loop. These two sites are conserved in the encapsidation signal of M1, a satellite RNA of the L-A virus.

23S RNA-derived replicon as a 'molecular tag' for monitoring inoculated wine yeast strains.

In this study we have developed a useful method to identify a particular yeast strain within a mixture of strains during must fermentation, based on the presence or absence of a stable genetic element derived from Saccharomyces cerevisiae 23S RNA autonomous replicon. 23S RNA is a natural virus‐like RNA replicon present in some S. cerevisiae strains, which encodes only its own RNA‐dependent RNA polymerase named p104. A modified version of 23S RNA (23S‐tagged RNA) was generated after transformation of S. cerevisiae wine strains with a launching plasmid, where six nucleotides were changed in the 23S RNA cDNA sequence without modifying the amino acid sequence of p104 RNA polymerase. Once generated, the 23S‐tagged RNA can replicate autonomously (without the plasmid), is very stable, is present in high copy number in stationary phase or nitrogen‐starved cells and confers no phenotype to the host, like the endogenous 23S RNA replicon. However, it can be distinguished from endogenous 23S RNA by reverse transcription followed by polymerase chain reaction (RT–PCR) with specific oligonucleotide primers. 23S RNA‐derived replicon can be used to tag wine yeast strains in order to monitor easily their prevalence over endogenous strains during wine fermentation. Copyright