Identification of an RNA Silencing Suppressor Encoded by a Symptomless Fungal Hypovirus, Cryphonectria Hypovirus 4.

Annisa Aulia,Nobuhiro Suzuki,Bradley I Hillman,Sakae Hisano,Kiwamu Hyodo,Hideki Kondo

doi:10.3390/biology10020100

Annisa Aulia, Nobuhiro Suzuki + Show 4 more

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https://doi.org/10.3390/biology10020100

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Abstract

Simple SummaryHost antiviral defense/viral counter-defense is an interesting topic in modern virology. RNA silencing is the primary antiviral mechanism in insects, plants, and fungi, while viruses encode and utilize RNA silencing suppressors against the host defense. Hypoviruses are positive-sense single-stranded RNA viruses with phylogenetic affinity to the picorna-like supergroup, including animal poliovirus and plant potyvirus. The prototype hypovirus Cryphonectria hypovirus 1, CHV1, is one of the best-studied fungal viruses. It is known to induce hypovirulence in the chestnut blight fungus, Cryphonectria parasitica, and encode an RNA silencing suppressor. CHV4 is another hypovirus asymptomatically that infects the same host fungus. This study shows that the N-terminal portion of the CHV4 polyprotein, termed p24, is a protease that autocatalytically cleaves itself from the rest of the viral polyprotein, and functions as an antiviral RNA silencing suppressor.Previously, we have reported the ability of a symptomless hypovirus Cryphonectria hypovirus 4 (CHV4) of the chestnut blight fungus to facilitate stable infection by a co-infecting mycoreovirus 2 (MyRV2)—likely through the inhibitory effect of CHV4 on RNA silencing (Aulia et al., Virology, 2019). In this study, the N-terminal portion of the CHV4 polyprotein, termed p24, is identified as an autocatalytic protease capable of suppressing host antiviral RNA silencing. Using a bacterial expression system, CHV4 p24 is shown to cleave autocatalytically at the di-glycine peptide (Gly214-Gly215) of the polyprotein through its protease activity. Transgenic expression of CHV4 p24 in Cryphonectria parasitica suppresses the induction of one of the key genes of the antiviral RNA silencing, dicer-like 2, and stabilizes the infection of RNA silencing-susceptible virus MyRV2. This study shows functional similarity between CHV4 p24 and its homolog p29, encoded by the symptomatic prototype hypovirus CHV1.

Highlights

We described an interesting virus/virus interplay in C. parasitica, in which a symptomless hypovirus, CHV4-C18 (Cryphonectria hypovirus 4, strain C-18), a member of the family Hypoviridae, facilitates the stable infection of a mycoreovirus, mycoreovirus 2 (MyRV2), which is susceptible to antiviral RNA silencing, and enhances its replication and its vertical transmission through asexual spores [28]
We show that the N-terminal portion of the CHV4-C18 polyprotein, termed p24, is a protease that autocatalytically cleaves itself from the rest of the viral polyprotein, and functions as an antiviral RNA silencing suppressor
An eGFPbased reporter system for monitoring dcl2 transcript levels was developed in the genetic background of standard C. parasitica strain EP155, in which the dcl2 promoter was linked to an egfp gene [39]

Summary

Introduction

There are interesting similarities and dissimilarities in antiviral RNA silencing among these fungi. The key enzyme genes associated with fungal, antiviral RNA silencing are generally transcriptionally upregulated by virus infection [2,4,11], while their induction magnitude is different between host fungi [7,12]. Specific fungal genes that are required for antiviral RNA silencing vary depending on fungal species. In C. parasitica (chestnut blight fungus), only one dicer-like (dcl2) and one argonaute-like (agl2) gene play central roles [1,2,13], and no RNA-dependent RNA polymerase (rdr) genes are required for antiviral RNA silencing [14], unlike in plants [15,16,17]. Additional dicer and agl genes contribute to antiviral RNA silencing [3,7,8]

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