Biological properties and expression strategy of rosellinia necatrix megabirnavirus 1 analysed in an experimental host, Cryphonectria parasitica

Abstract

Rosellinia necatrix megabirnavirus 1 (RnMBV1) with a bipartite dsRNA genome (dsRNA1 and dsRNA2) confers hypovirulence to its natural host, the white root rot fungus, and is thus regarded as a potential virocontrol (biocontrol) agent. Each segment has two large ORFs: ORF1 and partially overlapping ORF2 on dsRNA1 encode the major capsid protein (CP) and RNAdependent RNA polymerase (RdRp), whilst ORF3 and ORF4 on dsRNA2 encode polypeptides with unknown functions. Here, we report the biological and molecular characterization of this virus in the chestnut blight fungus, Cryphonectria parasitica, a filamentous fungus that has been used as a model for mycovirus research. Transfection with purified RnMBV1 particles into an RNAsilencing-defective strain (Ddcl-2) of C. parasitica and subsequent anastomosis with the WT strain (EP155) resulted in stable persistent infection in both host strains. However, accumulation levels in the two strains were different, being~20-fold higher in Ddcl-2 than in EP155. Intriguingly, whilst RnMBV1 reduced both virulence and growth rate in Ddcl-2, it attenuated virulence without affecting significantly other traits in EP155. Western blot analysis using antiserum against recombinant proteins encoded by either ORF1 or ORF2 demonstrated the presence of a 250 kDa protein in purified virion preparations, suggesting that RdRp is expressed as a CP fusion product via a "1 frameshift. Antiserum against the ORF3-encoded protein allowed the detection of 150, 30 and 23 kDa polypeptides specifically in RnMBV1-infected mycelia. Some properties of an RnMBV1 mutant with genome rearrangements, which occurred after transfection of Ddcl-2 and EP155, were also presented. This study provides an additional example of C. parasitica serving as a versatile, heterologous fungus for exploring virus-host interactions and virus gene expression strategies.

50-plus years of fungal viruses

“…dsRNA1 spans 8.9 kbp encompassing two partially overlapping ORFs on one strand. ORF1 encodes the CP and ORF2 encodes the RdRp, which is expressed as a fusion product with CP, probably via ribosomal frameshifting Salaipeth et al, 2014). dsRNA2 spans 7.2 kbp encompassing two non-overlapping ORFs in the same frame on one strand.…”

A novel single-stranded RNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix, with similarity to hypo-like viruses

“…Importantly, of these, Rosellinia necatrix megabirnavirus 1 (RnMBV1) and mycoreovirus 3 (MyRV3) attract attention because of their potential as virocontrol agents. Furthermore, many of them were shown to be able to infect a taxonomically distinct heterologous fungus, Cryphonectria parasitica , in which these viruses are targeted by RNA silencing, an anti-virus host defense (Kanematsu et al, 2010; Chiba et al, 2013a,b; Salaipeth et al, 2014). …”

Molecular characterization of a novel mycovirus from Rhizoctonia fumigata AG-Ba isolate C-314 Baishi

“…dsRNA1 and dsRNA2 of RnMBV1 were predicted to be encapsidated separately, and dsRNA1 could infect the fungal host independently. The ORF configuration of RnMBV1 dsRNA1 is similar to those of RfV1, PgLV-1 and LeV [31,32]. Based on the data presented above, we propose that RfV1, along with PgLV-1 and LeV, should be classified as members of a new viral genus related to the family Megabirnaviridae.…”

“…In RfV1, the sequence AAAUUUC was found at nt 5965-5971 immediately upstream of the stop codon of ORF1, which could act as the shifty heptamer. The -1 frameshift has been demonstrated for viruses with genome organizations similar to that of RfV1, such as the L-A dsRNA virus of Saccharomyces cerevisiae (ScV-L-A) [29], PgLV-1 [25,30], LeV [24] and dsRNA1 of Rosellinia necatrix megabirnavirus 1 (RnMBV1) [31]. The revised ORF2 (nt 5923-9873) encoded 1,315 aa, potentially yielding a 146.72-kDa putative protein (P2) containing the conserved motifs of the RNA-dependent RNA polymerase (RdRp).…”