Genome Organization of a New Double-Stranded RNA LA Helper Virus From Wine Torulaspora delbrueckii Killer Yeast as Compared With Its Saccharomyces Counterparts

Abstract

Wine killer yeasts such as killer strains of Torulaspora delbrueckii and Saccharomyces cerevisiae contain helper large-size (4.6 kb) dsRNA viruses (V-LA) required for the stable maintenance and replication of killer medium-size dsRNA viruses (V-M) which bear the genes that encode for the killer toxin. The genome of the new V-LA dsRNA from the T. delbrueckii Kbarr1 killer yeast (TdV-LAbarr1) was characterized by high-throughput sequencing (HTS). The canonical genome of TdV-LAbarr1 shares a high sequence identity and similar genome organization with its Saccharomyces counterparts. It contains all the known conserved motifs predicted to be necessary for virus translation, packaging, and replication. Similarly, the Gag-Pol amino-acid sequence of this virus contains all the features required for cap-snatching and RNA polymerase activity, as well as the expected regional variables previously found in other LA viruses. Sequence comparison showed that two main clusters (99.2–100% and 96.3–98.8% identity) include most LA viruses from Saccharomyces, with TdV-LAbarr1 being the most distant from all these viruses (61.5–62.5% identity). Viral co-evolution and cross transmission between different yeast species are discussed based on this sequence comparison. Additional 5′ and 3′ sequences were found in the TdV-LAbarr1 genome as well as in some newly sequenced V-LA genomes from S. cerevisiae. A stretch involving the 5′ extra sequence of TdV-LAbarr1 is identical to a homologous stretch close to the 5′ end of the canonical sequence of the same virus (self-identity). Our modeling suggests that these stretches can form single-strand stem loops, whose unpaired nucleotides could anneal to create an intramolecular kissing complex. Similar stem loops are also found in the 3′ extra sequence of the same virus as well as in the extra sequences of some LA viruses from S. cerevisiae. A possible origin of these extra sequences as well as their function in obviating ssRNA degradation and allowing RNA transcription and replication are discussed.

Yeast Interactions and Molecular Mechanisms in Wine Fermentation: A Comprehensive Review

“…Moreover, Ramírez et al [ 106 ] isolated and selected wine T. delbrueckii strains producing killer toxin Kbarr-1. This toxin is encoded by a dsRNA, TdV-Mbarr-1, structurally like M dsRNAs of S. cerevisiae , which both seem to be evolutionarily related [ 107 ].…”

New Insights into the Genome Organization of Yeast Double-Stranded RNA LBC Viruses

“…The slippery site for ribosomal frameshifting is "GGGUUUA" in V-LA, and "GGAUUUU" in V-LBC [8,9,[11][12][13]. Several cis stemloops located in the 3 -terminal region of LA (+)RNA are involved in its packaging and replication [3,8,9,[11][12][13][14][15]. The 3 -region of V-M (+)RNA (non-coding region) contains stemloops similar to the signals required for packaging and replication in L viruses.…”

“…This allows V-M to share the replication machinery with V-L for viral propagation. Moreover, the transcription initiation signal located at the 3 -end of canonical V-L (−)RNA (3 CTTTTT in V-LA and 3 CTTAAA in V-LBC; corresponding to 5 GAAAAA and 5 GAATTT in the (+)RNA strand, respectively), is also mimicked in the (−)RNA 3 -end of V-M canonical sequence [4,6,9,14,[16][17][18][19].…”

“…In some cases, these extra ssRNA stretches can form stem-loops, whose unpaired nucleotides could form intramolecular kissing complexes. It has been suggested that these extra sequences may form complex RNA structures preserving the virus ssRNA from degradation and facilitating the dsRNA synthesis [14]. Amino-acid Gag-Pol sequences of LA viruses share great identity (87-99%).…”

“…Despite this, a 42-amino-acid variable sequence stretch (42-58% identity) has been described, which is located between Gag and Pol domains. This low identity stretch (LIS) does not seem to be of much importance for virus replication, and its function could simply be the separation of Gag and Pol domains in the Gag-Pol fusion protein [14,15]. New V-LBC genomes from Sc and Td yeasts have recently been sequenced using HTS techniques and subsequently characterized.…”