Discovery of divided RdRp sequences and a hitherto unknown genomic complexity in fungal viruses

Abstract

By identifying variations in viral RNA genomes, cutting-edge metagenome technology has potential to reshape current concepts about the evolution of RNA viruses. This technology, however, cannot process low-homology genomic regions properly, leaving the true diversity of RNA viruses unappreciated. To overcome this technological limitation, we applied an advanced method, Fragmented and Primer-Ligated Double-stranded (ds) RNA Sequencing (FLDS), to screen RNA viruses from 155 fungal isolates, which allowed us to obtain complete viral genomes in a homology-independent manner. We created a high-quality catalog of 19 RNA viruses (12 viral species) that infect Aspergillus isolates. Among them, nine viruses were not detectable by the conventional methodology involving agarose gel electrophoresis of dsRNA, a hallmark of RNA virus infections. Segmented genome structures were determined in 42 per cent of the viruses. Some RNA viruses had novel genome architectures; one contained a dual methyltransferase domain and another had a separated RNA-dependent RNA polymerase (RdRp) gene. A virus from a different fungal taxon (Pyricularia) had an RdRp sequence that was separated on different segments, suggesting that a divided RdRp is widely present among fungal viruses, despite the belief that all RNA viruses encode RdRp as a single gene. These findings illustrate the previously hidden diversity and evolution of RNA viruses, and prompt reconsideration of the structural plasticity of RdRp.

Keywords: Aspergillus fumigatus; FLDS; RNA virus; RdRp; viral genome.

Figure 1.

Detection of dsRNA by agarose gel electrophoresis. Comprehensive screening of 155 Aspergillus strains produced nine strains with positive virus-like bands. The dsRNAs from nine strains (four from A. fumigatus and five of A. lentulus) were electrophoresed, and the gel was stained with GelRed. Lanes: M, DNA marker; 1, IFM 62632; 2, IFM 63147; 3, IFM 63431; 4, IFM 64916; 5, IFM 62627; 6, IFM 64004; 7, IFM 64003; 8, IFM 63547; and 9, IFM 65052.

Figure 2.

Characterization of AfuRV1. (A) The RNA genome structure model for AfuRV1. The predicted ORFs are indicated by white boxes. The domains identified as methyl transferase, RdRP, and helicase are indicated in yellow, blue and brown boxes, respectively. Molecular phylogenetic analysis on the RdRp (B), methyltransferase (C), and helicase (D) domains was performed by maximum likelihood-based methodology. The numbers indicate the percentage bootstrap support from 1,000 RAxML bootstrap replicates. The best-fitting amino acid substitution models were [LG + F + G] (B) (C) and [rtREV + F + G] (D). The accession numbers and full virus names are listed in Table S4. The scale bar represents the number of substitutions per site. The viral sequences from fungi and invertebrates are indicated by orange circles or blue squares, respectively. AfuRV1 sequences are shown in red font.

Figure 3.

Phylogenetic tree for the RdRp from the Narnaviridae family. The RdRp amino acid sequences without motifs C and D from AleNV1, AfuNV2, MoNV1, and MoNV2 (without the C/D motif is shown in a gray box) and related viruses were used to construct the phylogenetic tree. The numbers indicate the percentage bootstrap support from 1,000 RAxML bootstrap replicates. The best-fitting amino acid substitution model was [LG + F + G]. The accession numbers and full virus names are listed in Table S4. The viral sequences from fungi, invertebrates, oomycetes, plants, and an unknown host are indicated by orange circles, blue squares, light blue diamonds, green triangles, and question marks, respectively. The viral sequences identified in this study are shown in red font.

Figure 4.

Characterization of AfuNV2 and MoNV1. (A) RNA genome structure model for AfuNV2. The predicted ORFs are represented by boxes, and the first identified RdRp domain is shown by a blue box. (B) Schematic model of RdRp protein with conserved motifs in AfuNV2, MoNV1, and ScNV-20S genomes. Conserved A–D and F motifs in RdRp from ScNV-20S, a type strain of narnavirus, are shown (Venkataraman et al. 2018; Jia and Gong 2019). (C) Multiple alignment of the deduced amino acid sequences of the RdRp motifs for the Narnavirus genus. Among the 18–24 sequences, the amino acid positions with 100 per cent matches and >50 per cent matches are depicted by green and gray shading, respectively. Dominant amino acid residues at positions with >50 per cent amino acid matches are shown in bold. The number of amino acids not shown in the alignment is noted in each sequence. The accession numbers and full virus names are listed in Table S4. (D) RNA genome structure model for MoNV1. The predicted ORFs are represented by boxes, and the first identified RdRp domain is shown by a blue box.

Figure 5.

Homology modeling. (A) RdRp from bacteriophage Qβ and AfuNV2 virus. Gray cartoon shows three-dimensional structure of the template (RdRp from bacteriophage Qβ). Green and blue cartoon show the model structure of ORF1 and ORF2 (green, ORF1; blue, ORF2). (B) The arrangements of the amino acid residues of motifs A and C. The green and blue stick model show amino acid residues of motifs A and C from AfuNV2, respectively (red, oxygen atom; blue, nitrogen atom). White stick model shows the corresponding residues from bacteriophage Qβ. Characters refer to amino acid residues of AfuNV2 and parentheses are amino acid residues of bacteriophage Qβ. (C) Hydrogen bonds between ORF1 and ORF2. Green and blue ball models show ORF1 and ORF2 residues, respectively. Stick models show amino acid residues of ORF1 and ORF2 that interact with each other. Pink dashed lines refer to hydrogen bond between amino acid residues of ORF1 and ORF2.