Nucleotide sequence of Zygosaccharomyces bailii virus Z: Evidence for +1 programmed ribosomal frameshifting and for assignment to family Amalgaviridae

Abstract

Zygosaccharomyces bailii virus Z (ZbV-Z) is a monosegmented dsRNA virus that infects the yeast Zygosaccharomyces bailii and remains unclassified to date despite its discovery >20 years ago. The previously reported nucleotide sequence of ZbV-Z (GenBank AF224490) encompasses two nonoverlapping long ORFs: upstream ORF1 encoding the putative coat protein and downstream ORF2 encoding the RNA-dependent RNA polymerase (RdRp). The lack of overlap between these ORFs raises the question of how the downstream ORF is translated. After examining the previous sequence of ZbV-Z, we predicted that it contains at least one sequencing error to explain the nonoverlapping ORFs, and hence we redetermined the nucleotide sequence of ZbV-Z, derived from the same isolate of Z. bailii as previously studied, to address this prediction. The key finding from our new sequence, which includes several insertions, deletions, and substitutions relative to the previous one, is that ORF2 in fact overlaps ORF1 in the +1 frame. Moreover, a proposed sequence motif for +1 programmed ribosomal frameshifting, previously noted in influenza A viruses, plant amalgaviruses, and others, is also present in the newly identified ORF1–ORF2 overlap region of ZbV-Z. Phylogenetic analyses provided evidence that ZbV-Z represents a distinct taxon most closely related to plant amalgaviruses (genus Amalgavirus, family Amalgaviridae). We conclude that ZbV-Z is the prototype of a new species, Zygosaccharomyces bailii virus Z, which we propose to assign as type species of a new genus of monosegmented dsRNA mycoviruses in family Amalgaviridae. Comparisons involving other unclassified mycoviruses with RdRps apparently related to those of plant amalgaviruses, and having either mono- or bisegmented dsRNA genomes, are also discussed.

Amalga-like virus infecting Antonospora locustae , a microsporidian pathogen of grasshoppers, plus related viruses associated with other arthropods

“…Purified spores of A. locustae isolate ATCC30860 were purchased from M&R Durango, Inc. Total RNAs were purified from these spores using previously described methods (Depierreux et al, 2016), though with some modifications. Approximately 1e+8 spores were subjected to vortexing with 0.25 g glass beads and 8 U zymolase (Zymo Research) at room temperature for 1 h. Following disruption of the spore cell walls, samples were mixed with 500 μL TRIzol reagent (Ambion) and 100 μL chloroform.…”

“…Total dsRNAs were enriched from total RNA samples using the microcrystalline cellulose method (Castillo et al, 2011), largely as described by Depierreux et al, (2016). To confirm the chemical nature of the enriched dsRNAs, DNase I and RNase A treatments were used.…”

“…Recently, we have redetermined and amended the complete genome sequence of ZbV-Z from Z. bailii 412, providing further support for its relationship to plant amalgaviruses (Depierreux et al, 2016). We have additionally proposed ZbV-Z/412 as prototype strain of a new genus, “Zybavirus”, in family Amalgaviridae .…”

Molecular Identification and Characterization of Two Rubber Dandelion Amalgaviruses

“…Translation resolves on the advanced ribosome in the +1 frame (Figure 1.B). This phenomenon has been predicted to be widespread among most plant amalgaviruses (Nibert et al, 2016). RdLV1 & RdLV2 share a 55.9 % genome nt identity and a 49.5 % aa pairwise identity between their predicted RdRPs.…”

“…The structural highlights of RdLV1 & RdLV2 were compared to the ICTV recognized Amalgavirus species (Table 1) (Depierreux et al, 2016), is consistently low, supporting that both RdLV1 & RdLV2 could be members of the Amalgavirus genus.…”