The Characterization of a Novel Virus Discovered in the Yeast Pichia membranifaciens.

Mycoviruses are widely distributed across fungi, including the yeasts of the Saccharomycotina subphylum. This manuscript reports the first double-stranded RNA (dsRNA) virus isolated from Pichia membranifaciens. This novel virus has been named Pichia membranifaciens virus L-A (PmV-L-A) and is a member of the Totiviridae. PmV-L-A is 4579 bp in length, with RNA secondary structures similar to the packaging, replication, and frameshift signals of totiviruses that infect Saccharomycotina yeasts. PmV-L-A was found to be part of a monophyletic group within the I-A totiviruses, implying a shared ancestry between mycoviruses isolated from the Pichiaceae and Saccharomycetaceae yeasts. Energy-minimized AlphaFold2 molecular models of the PmV-L-A Gag protein revealed structural conservation with the Gag protein of Saccharomyces cerevisiae virus L-A (ScV-L-A). The predicted tertiary structure of the PmV-L-A Pol and other homologs provided a possible mechanism for totivirus RNA replication due to structural similarities with the RNA-dependent RNA polymerases of mammalian dsRNA viruses. Insights into the structure, function, and evolution of totiviruses gained from yeasts are essential because of their emerging role in animal disease and their parallels with mammalian viruses.

Cap snatching in yeast L-BC double-stranded RNA totivirus.

Yeast L-A double-stranded RNA virus furnishes its transcript with a 5' cap structure by a novel cap-snatching mechanism in which m(7)Gp from a host mRNA cap structure is transferred to the 5'-diphosphate terminus of the viral transcript. His-154 of the coat protein Gag forms an m(7)Gp adduct, and the H154R mutation abolishes both m(7)Gp adduct formation and cap snatching. Here we show that L-BC, another totivirus closely related to L-A, also synthesizes 5'-diphosphorylated transcripts and transfers m(7)Gp from mRNA to the 5' termini of the transcripts. L-BC Gag also covalently binds to the cap structure and the mutation H156R, which corresponds to H154R of L-A Gag, abolishes cap adduct formation. Cap snatching of the L-BC virus is very similar to that of L-A; N7 methylation of the mRNA cap is essential for cap donor activity, and only 5'-diphosphorylated RNA is used as cap acceptor. L-BC cap snatching is also activated by viral transcription. Furthermore, both viruses require Mg(2+) and Mn(2+) for cap snatching. These cations are not only required for transcription activation but also directly involved in the cap transfer process. These findings support our previous proposal that the cap-snatching mechanism of the L-A virus is shared by fungal totiviruses closely related to L-A. Interestingly, L-A and L-BC viruses accept either viral transcript as cap acceptor in vitro. Because L-A and L-BC viruses cohabit in many yeast strains, it raises the possibility that their cohabitation in the same host may be beneficial for their mutual cap acquisition.

L-A-lus, a new variant of the L-A totivirus found in wine yeasts with Klus killer toxin-encoding Mlus double-stranded RNA: possible role of killer toxin-encoding satellite RNAs in the evolution of their helper viruses.

Yeast killer viruses are widely distributed in nature. Several toxins encoded in double-stranded RNA (dsRNA) satellites of the L-A totivirus have been described, including K1, K2, K28, and Klus. The 4.6-kb L-A genome encodes the Gag major structural protein that forms a 39-nm icosahedral virion and Gag-Pol, a minor fusion protein. Gag-Pol has transcriptase and replicase activities responsible for maintenance of L-A (or its satellite RNAs). Recently we reported a new killer toxin, Klus. The L-A virus in Klus strains showed poor hybridization to known L-A probes, suggesting substantial differences in their sequences. Here we report the characterization of this new L-A variant named L-A-lus. At the nucleotide level, L-A and L-A-lus showed only 73% identity, a value that increases to 86% in the amino acid composition of Gag or Gag-Pol. Two regions in their genomes, however, the frameshifting region between Gag and Pol and the encapsidation signal, are 100% identical, implying the importance of these two cis signals in the virus life cycle. L-A-lus shows higher resistance than L-A to growth at high temperature or to in vivo expression of endo- or exonucleases. L-A-lus also has wider helper activity, being able to maintain not only Mlus but also M1 or a satellite RNA of L-A called X. In a screening of 31 wine strains, we found that none of them had L-A; they carried either L-A-lus or a different L-A variant in K2 strains. Our data show that distinct M killer viruses are specifically associated with L-As with different nucleotide compositions, suggesting coevolution.

Cap-snatching mechanism in yeast L-A double-stranded RNA virus.

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 ß-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.

Ded1p, a conserved DExD/H-box translation factor, can promote yeast L-A virus negative-strand RNA synthesis in vitro.

Viruses are intracellular parasites that must use the host machinery to multiply. Identification of the host factors that perform essential functions in viral replication is thus of crucial importance to the understanding of virus-host interactions. Here we describe Ded1p, a highly conserved DExD/H-box translation factor, as a possible host factor recruited by the yeast L-A double-stranded RNA (dsRNA) virus. We found that Ded1p interacts specifically and strongly with Gag, the L-A virus coat protein. Further analysis revealed that Ded1p interacts with the L-A virus in an RNA-independent manner and, as a result, L-A particles can be affinity purified via this interaction. The affinity-purified L-A particles are functional, as they are capable of synthesizing RNA in vitro. Critically, using purified L-A particles, we demonstrated that Ded1p specifically promotes L-A dsRNA replication by accelerating the rate of negative-strand RNA synthesis in vitro. In light of these data, we suggest that Ded1p may be a part of the long sought after activity shown to promote yeast viral dsRNA replication. This and the fact that Ded1p is also required for translating brome mosaic virus RNA2 in yeast thus raise the intriguing possibility that Ded1p is one of the key host factors favored by several evolutionarily related RNA viruses, including the human hepatitis C virus.