Mycoreovirus genome rearrangements associated with RNA silencing deficiency.

Ana Eusebio-Cope,Nobuhiro Suzuki

doi:10.1093/nar/gkv239

Abstract

Mycoreovirus 1 (MyRV1) has 11 double-stranded RNA genome segments (S1 to S11) and confers hypovirulence to the chestnut blight fungus, Cryphonectria parasitica. MyRV1 genome rearrangements are frequently generated by a multifunctional protein, p29, encoded by a positive-strand RNA virus, Cryphonectria hypovirus 1. One of its functional roles is RNA silencing suppression. Here, we explored a possible link between MyRV1 genome rearrangements and the host RNA silencing pathway using wild-type (WT) and mutant strains of both MyRV1 and the host fungus. Host strains included deletion mutants of RNA silencing components such as dicer-like (dcl) and argonaute-like (agl) genes, while virus strains included an S4 internal deletion mutant MyRV1/S4ss. Consequently, intragenic rearrangements with nearly complete duplication of the three largest segments, i.e. S1, S2 and S3, were observed even more frequently in the RNA silencing-deficient strains Δdcl2 and Δagl2 infected with MyRV1/S4ss, but not with any other viral/host strain combinations. An interesting difference was noted between genome rearrangement events in the two host strains, i.e. generation of the rearrangement required prolonged culture for Δagl2 in comparison with Δdcl2. These results suggest a role for RNA silencing that suppresses genome rearrangements of a dsRNA virus.

Highlights

RNA viruses are prone to recombination, which is one of the driving forces of virus molecular diversity and evolution [1,2,3,4,5]
Disruption of the rdr1 gene was confirmed by Southern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses (Supplementary Figure S2 B and C)
The rdr1 gene was replaced with a gene cassette encoding hygromycin B phosphotransferase, which confers resistance to hygromycin; neither of the rdr1-specific DNA bands was detected by Southern blotting or RT-PCR analysis

Summary

Introduction

RNA viruses are prone to recombination, which is one of the driving forces of virus molecular diversity and evolution [1,2,3,4,5]. RNA recombinants have long been utilized for studies of viral RNA replication, which have led to the identification of RNA sequences and protein elements important for RNA synthesis, virion assembly and symptom expression. This is true for members of the family Reoviridae, where reverse genetics is mostly unavailable [11,12]. The Reoviridae is one of the largest families of double-stranded (ds) RNA viruses and includes members infecting a wide range of eukaryotic organisms (fungi, plants and animals). The development of reverse genetics for animal reoviruses such as orthoreoviruses (helper virus-independent) [14] and rotaviruses (helper virus-dependent) [15,16,17] has led to the creation of artificial replicable genome segments that retain authentic essential terminal sequences [18,19]

Methods

Results

Conclusion