Novel viruses of the family Partitiviridae discovered in Saccharomyces cerevisiae

Abstract

It has been 49 years since the last discovery of a new virus family in the model yeast Saccharomyces cerevisiae. A large-scale screen to determine the diversity of double-stranded RNA (dsRNA) viruses in S. cerevisiae has identified multiple novel viruses from the family Partitiviridae that have been previously shown to infect plants, fungi, protozoans, and insects. Most S. cerevisiae partitiviruses (ScPVs) are associated with strains of yeasts isolated from coffee and cacao beans. The presence of partitiviruses was confirmed by sequencing the viral dsRNAs and purifying and visualizing isometric, non-enveloped viral particles. ScPVs have a typical bipartite genome encoding an RNA-dependent RNA polymerase (RdRP) and a coat protein (CP). Phylogenetic analysis of ScPVs identified three species of ScPV, which are most closely related to viruses of the genus Cryspovirus from the mammalian pathogenic protozoan Cryptosporidium parvum. Molecular modeling of the ScPV RdRP revealed a conserved tertiary structure and catalytic site organization when compared to the RdRPs of the Picornaviridae. The ScPV CP is the smallest so far identified in the Partitiviridae and has structural homology with the CP of other partitiviruses but likely lacks a protrusion domain that is a conspicuous feature of other partitivirus particles. ScPVs were stably maintained during laboratory growth and were successfully transferred to haploid progeny after sporulation, which provides future opportunities to study partitivirus-host interactions using the powerful genetic tools available for the model organism S. cerevisiae.

Fig 1. The identification of PVs in S. cerevisiae.

(A) Virus contigs were assembled from the sequencing of dsRNAs extracted from 520 strains of S. cerevisiae. (B) Agarose gel electrophoresis analysis of dsRNAs extracted from two PV-infected strains of S. cerevisiae. (C) DsRNAs extracted in panel B were subjected to incubation at 37°C with and without RNase III, and the resulting products were analyzed by agarose gel electrophoresis. (D) Two-step RT-PCR of total nucleic acids extracted from S. cerevisiae targeting the CP or RdRP genes with (+RT) or without (-RT) reverse transcriptase. Strain BJH001 was used as a negative control as it does not harbor PVs. Primers targeting the yeast gene UBC6 were used as a PCR-positive control for genomic DNA.

Fig 2. The presence of ScPVs is independent of satellite dsRNAs and totiviruses.

(A) Top Schematic of a killer toxin production by a killer yeast strain with a zone of growth inhibition. Bottom Killer activity of strain CYC1172 before and after treatment with cycloheximide (B) Electrophoretic mobilities of dsRNAs extracted from CYC1172 before and after treatment with cycloheximide. (C) Agarose gel electrophoresis of dsRNAs extracted from selected strains of S. cerevisiae that contained either a totivirus and PV (lane 1) or only a PV (lanes 2–5).

Fig 3

ScPVs are closely related to C. parvum PVs (A) Strains of S. cerevisiae isolated from different sources and the proportion infected with ScPVs (dotted regions). (B) Comparing the protein lengths of RdRPs and CPs of diverse PVs to ScPV1-5509, SCPV2-858, and ScPV3-1172. (C) A PhyML maximum likelihood phylogenetic model of the relatedness of PVs based on the amino acid sequence of the RdRP [50]. The RdRP from the picobirnavirus OaPV was used as an outgroup. The VT amino-acid exchange rate matrices were selected with a gamma distribution (G), a proportion of invariable sites (I), and empirical amino acid frequencies in the alignment fit these data best, as judged by PROTTEST [51]. The numbers at each node are the bootstrap values from 1000 iterations. The scale bar represents the distance of one amino acid substitution per site. The amino acid sequences in the phylogeny are from the viruses listed in S7 Table.

Fig 4. Genome organization of PVs from S. cerevisiae.

(A) Schematic representation of dsRNA1 and dsRNA2 of three species of PV from S. cerevisiae as well as CSpV1. The ORFs are represented as rectangles that encode the RdRP and CP. Stem-loop structures are annotated to represent similar structures in the 5’ UTRs of each species (S3 Fig). ^#/*/**/*** represent the pairs of CSE sequences in the terminal 3’ UTRs (B) Secondary structure of RNA sequence present in the 3’ CSE of dsRNA1 and dsRNA2 of ScPV1-5509, ScPV2-858, and ScPV3-1172.

Fig 5. The ScPV CP and RdRP share structural similarities with PVs and poliovirus.

(A) Visualization of ScPV1 particles by TEM as indicated by arrowheads. Scale bars are 500 nm (main image) and 25 nm (inset). (B) Molecular modeling using AlphaFold2 and energy minimization of the S-domain of three species of ScPV and CSpV1 compared to the solved structure of PCV1 (PDB:7ncr). The α-helices (blue rectangles) and β-sheets (magenta arrows) are labeled according to their common positioning within the S-domains. (C) An overlay of the five structures represented in panel B. (D) Molecular models of CSpV1, ScPV1, ScPV2, and ScPV3 RdRPs generated by comparison to poliovirus 1 RdRP (PDB:1ra6). Red, fingers domain; Blue, thumb domain; White, palm domain. (E) A superimposition of the RdRP conserved catalytic motifs A-F of poliovirus 1, CSpV1, ScPV1, ScPV2, and ScPV3. Red, motif A; Blue, motif B; Green, motif C; Purple, motif D; Cyan, motif E; Yellow, motif F.