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.

The 5′ cap structure (m ⁷ GpppX-) is an essential feature of eukaryotic mRNA required for mRNA stability and efficient translation. Influenza virus furnishes its mRNA with this structure by a cap-snatching mechanism, in which the viral polymerase cleaves host mRNA endonucleolytically 10–13 nucleotides from the 5′ end and utilizes the capped fragment as a primer to synthesize viral transcripts. Here we report a unique cap-snatching mechanism by which the yeast double-stranded RNA totivirus L-A furnishes its transcript with a cap structure derived from mRNA. Unlike influenza virus, L-A transfers only m ⁷ Gp from the cap donor to the 5′ end of the viral transcript, thus preserving the 5′ α- and β-phosphates of the transcript in the triphosphate linkage of the final product. This in vitro capping reaction requires His154 of the coat protein Gag, a residue essential for decapping of host mRNA and known to form m ⁷ Gp-His adduct. Furthermore, the synthesis of capped viral transcripts in vivo and their expression were greatly compromised by the Arg154 mutation, indicating the involvement of Gag in the cap-snatching reaction. The overall reaction and the structure around the catalytic site in Gag resemble those of guanylyltransferase, a key enzyme of cellular mRNA capping, suggesting convergent evolution. Given that Pol of L-A is confined inside the virion and unable to access host mRNA in the cytoplasm, the structural protein Gag rather than Pol catalyzing this unique cap-snatching reaction exemplifies the versatility as well as the adaptability of eukaryotic RNA viruses.

Most strains of Saccharomyces cerevisiae harbor L-A double-stranded RNA (dsRNA), 4.5 kilobases long, contained in virus-like particles (VLPs). These L-A VLPs can be separated by CsCl density gradient centrifugation into a main peak of particles, containing full-length L-A dsRNA, which synthesizes only plus-strand single-stranded RNA (ssRNA), and a lighter fraction of VLPs, containing plus-strand ssRNA, which has L-A dsRNA-synthesizing activity. This dsRNA-synthesizing activity was present in particles from logarithmically growing cells but not from stationary-phase cells. The newly synthesized strand of dsRNA in the lightest particles was full-length minus strand. All or almost all of the new minus strand was synthesized in vitro, and the rate of chain elongation was approximately 100 nucleotides per minute. The lightest particles synthesized plus-strand ssRNA only after completion of dsRNA synthesis, indicating that the same particle contains dsRNA- and ssRNA-synthesizing enzyme(s). We also observed dsRNA-synthesizing activity in L-BC dsRNA-containing particles similar to that in L-A VLPs.

Narnavirus 23S RNA is a persistent positive-stranded RNA virus found in yeast Saccharomyces cerevisiae . The viral genome (2.9 kb) only encodes its RNA-dependent RNA polymerase, p104. Here we report the generation of 23S RNA virus, with high frequency, from a vector containing the entire viral cDNA sequence. When the conserved GDD (Gly-Asp-Asp) motif of RNA-dependent RNA polymerase was modified, the vector failed to generate the virus, indicating that an active p104 is essential for replication. Successful launching required transcripts having the proper viral 3′ terminus generated in vivo . This was accomplished through in vivo processing of the primary transcripts by the hepatitis delta virus antigenomic ribozyme directly fused to the 3′ terminus of the 23S RNA genome. Although the primary transcripts also contained extra nucleotides at their 5′ ends derived from the vector, the launched virus possessed the authentic 5′ terminus of the viral genome without these extra nucleotides. Modifications of the genome sequence at the 5′ and 3′ termini abolished viral generation, indicating that the viral genome has cis-acting signals for replication at both termini. The great ease to generate the virus will facilitate the identification of these cis-acting signals. Furthermore, the virus, once generated, can be transmitted to daughter cells indefinitely without the vector or any selection, which makes the 23S RNA virus-launching system particularly useful for investigating the basis for RNA virus persistence.