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. 2015 Jul 21;112(29):9100-5.
doi: 10.1073/pnas.1419225112. Epub 2015 Jul 2.

A novel mycovirus from Aspergillus fumigatus contains four unique dsRNAs as its genome and is infectious as dsRNA

Lakkhana Kanhayuwa  1 , Ioly Kotta-Loizou  1 , Selin Özkan  1 , A Patrick Gunning  2 , Robert H A Coutts  3
Affiliations

A novel mycovirus from Aspergillus fumigatus contains four unique dsRNAs as its genome and is infectious as dsRNA

Lakkhana Kanhayuwa et al. Proc Natl Acad Sci U S A. .

Abstract

We report the discovery and characterization of a double-stranded RNA (dsRNA) mycovirus isolated from the human pathogenic fungus Aspergillus fumigatus, Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1), which reveals several unique features not found previously in positive-strand RNA viruses, including the fact that it represents the first dsRNA (to our knowledge) that is not only infectious as a purified entity but also as a naked dsRNA. The AfuTmV-1 genome consists of four capped dsRNAs, the largest of which encodes an RNA-dependent RNA polymerase (RdRP) containing a unique GDNQ motif normally characteristic of negative-strand RNA viruses. The third largest dsRNA encodes an S-adenosyl methionine-dependent methyltransferase capping enzyme and the smallest dsRNA a P-A-S-rich protein that apparently coats but does not encapsidate the viral genome as visualized by atomic force microscopy. A combination of a capping enzyme with a picorna-like RdRP in the AfuTmV-1 genome is a striking case of chimerism and the first example (to our knowledge) of such a phenomenon. AfuTmV-1 appears to be intermediate between dsRNA and positive-strand ssRNA viruses, as well as between encapsidated and capsidless RNA viruses.

Keywords: capping enzyme; infectious dsRNA; mycovirus; protoplast transfection; virus evolution.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Genomic characteristics and organization of Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1). (A) AfuTMV-1 was purified from Af293 mycelia, and dsRNA isolated using phenol/chloroform was fractionated on a 1% agarose gel, and the positions of dsRNAs 1–4 in lane 1 are shown. Lane M contains HyperLadder I kb DNA marker. (B) RNase III sensitivity of purified AfuTmV-1 and AfuTmV-1 dsRNA was investigated, respectively, and untreated samples of each are shown following agarose gel electrophoresis in lanes 1 and 3, with RNase III digestions of each shown in lanes 2 and 4. (C) Genome organization of AfuTMV-1 dsRNAs 1–4 showing putative ORFs and UTRs. The positions of motifs characteristic for RdRP and methyltransferase (SAM) are shown as gray boxes on dsRNA 1 and dsRNA 3, respectively. (D) Assignation of cDNA clones specific to dsRNAs 1–4 by northern hybridization. Purified viral dsRNAs were fractionated by electrophoresis in 1% agarose gel in 1× TAE, denatured, blotted onto nylon membrane, and probed with clones specific for each dsRNA as shown in C. Lane 1 shows the AfuTmV-1 dsRNA profile, and lanes 2–5, individual transfers hybridized with probes specific for dsRNAs 1–4, respectively.
Fig. 2.
Fig. 2.
Viral dsRNAs extracted from transfected cultures, RT-PCR amplification and northern detection of ssRNA and dsRNA from A. fumigatus isolate NK125 following transfection with AfuTmV-1. (A) AfuTmV-1 dsRNAs extracted from transfected cultures (ATV-1 and ATR-1), wild-type (Af293) and virus-free (NK125) isolates. (B) RT-PCR amplification of a 337-bp segment from dsRNA 2. Lane M contains HyperLadder I kb DNA marker. (C) Amounts of viral ssRNA and dsRNA, equivalent to 10 mg of total nucleic acids before LiCl fractionation, were electrophoresed in 1.0% nondenaturing agarose gels and blotted onto nylon membranes. The samples were treated with DNase I (lanes 1, 3, 5, and 7) or DNase I and S1 nuclease (lanes 2, 4, 6, and 8). Hybridization was carried out using positive-strand specific and negative-strand specific riboprobes for all four dsRNAs. The positions of the single-stranded forms of each RNA are indicated on the blots by arrows. RT-PCR amplification of a 467-bp segment from dsRNA 1 (D) and a 337-bp segment from dsRNA 2 (E) from equal amounts of RNA extracted from transfected cultures (ATV-1, ATRS1 nuclease, and ATRRNase III), wild-type (Af293) and virus-free (NK125) isolates. Lane M contains GeneRuler 100-bp DNA ladder.
Fig. 3.
Fig. 3.
AFM images of purified AfuTmV-1. AfuTmV-1 is visualized as numerous chain-like, linear nucleic acids of different lengths corresponding to those predicted from the genomic size of the four genomic dsRNAs. In the middle panel, arrows indicate the AfuTmV-1 PAS-rich ORF 4-encoded protein putatively associated with dsRNA 1.
See this image and copyright information in PMC

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