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

Abstract

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.

Keywords: Cryphonectria parasitica; Dicer; RNA silencing; RNAi suppressor; chestnut blight fungus; co-infection; hypovirus; mycovirus; reovirus.

Figure 1

CHV4 suppresses antiviral RNA silencing via inhibiting dcl2 transcriptional upregulation. (A) The organization of a reporter constructs pCPXHY-C18-dcl2pro::egfp. An egfp gene fused with a 2.2 kbp C. parasitica C18 genomic sequence containing the dcl2 promoter region was cloned into pCPXHY1 and used to transform a virus-free isogenic isolate of C. parasitica C18 strain (C18-VF) [28]. (B,C) CHV4-mediated suppression of the GFP reporter induction. The reporter fungal strain with pCPXHY-C18-dcl2pro::egfp (C18/dcl2pro-eGFP) was infected by CHV1-∆p69 and MyRV2 (B), which are strong dcl2 triggers, or CHV4 and CHV1 wild type (C). Values in the respective panels of (B) show the relative intensity of the reporter eGFP green fluorescence quantified by ImageJ, with the CHV1-∆p69-infected strain expressed as 100. Total RNA fractions were obtained from two biological replicates of the reporter fungal strain (C18/dcl2pro-eGFP) infected by each virus, and subjected to northern blotting to monitor dcl2 and egfp transcript levels. Hybridization and probe preparation are described in the Materials and Method section. Mean values of band intensity in the northern blots (C) quantified by ImageJ are shown below each blot. RT-PCR detection for CHV4 infection or dsRNA electrophoretic gel analysis for MyRV2 (viral dsRNA genome) or CHV1 and CHV1-∆p69 (viral replicative dsRNA form) infection in the reporter fungal strain were conducted. Ribosomal RNA (28S rRNA) was used as a loading control.

Figure 2

Viral-derived small RNA profiling of C. parasitica C18 strain in either singly or doubly infected by CHV4-C18 and MyRV2. (A) Sense and small antisense RNAs (15 to 32 nt) derived from singly or doubly infected CHV4-C18 and MyRV2 are shown on a per-million-total-small-RNA read (the y-axis represents the one-thousandth read numbers). The C18 strain infected singly by CHV1-EP713 (wild type) was also included for the analysis as a reference. The percentage shown on each bar represents the ratio of negative strand small RNAs. (B) Size distributions of small RNAs derived from CHV1 in singly infected C18. (C) Size distributions of virus-derived small RNAs in single and double infections of strain C18 by CHV4-C18 and/or MyRV2. Blue and red bars denote plus-strand and minus-strand small RNAs (B,C).

Figure 3

Autocatalytic proteolytic activity of CHV4 p24 encoded at the most N terminal portion of the viral polyprotein. (A) Schematic representation of the N terminal portion of the CHV4-C18 polyprotein. The N-terminal coding regions spanning amino acids 1 to 473 (the estimated molecular weight of ~ 52 kDa) was cloned into the KpnI-NdeI site of an Escherichia coli expression vector, pCold. The 52kDa coding region was fused in frame with a His-tag and GST at the N-terminus and an HA-tag at the C-terminus (pCold-His/GST-CHV4 52kDa-HA, 86 kDa). The predicted cysteine and histidine residues and di-glycine are shown on the top. (B) The self-cleavage activity of CHV4-C18 p24 in the recombinant E. coli cells. Total proteins in E. coli cells transformed by the CHV4-52kDa expression construct and empty vector were electrophoresed in SDS-PAGE gel and stained with Coomassie Brilliant Blue. Two protein bands of 52kDa and 28 kDa were specifically induced in the recombinant cells with the CHV4-52kDa construct upon induction (left panel, shown with arrows). Unpurified fractions of the over-expressed protein encoded by the N terminal portion of CHV4 were blotted onto PVDF membrane, and the 28 kDa protein band was subjected to chemical sequencing. The six determined amino acid residues were the same as those deduced from the CHV4 nucleotide sequence. The cleavage site was experimentally identified as Gly245↓Gly246, which had been predicted by Linder-Basso et al. [41].

Figure 4

Suppression of transcriptional upregulation of dcl2 by CHV4-C18 p24. Total RNA fractions were obtained from fungal strains shown on the top. C. parasitica C18 non-transformants infected by MyRV2 (C18/MyRV2), and MyRV2 plus CHV4 (C18/MyRV2+CHV4), CHV4 p24 transformants uninfected (C18p24-VF) or infected by MyRV2 (C18p24/MyRV2). Four independent transformants (1 to 5) were tested in this experiment. Total RNA was examined by northern blotting for dcl2 transcripts, MyRV2 S10 mRNA, and CHV4-C18 p24 transgene transcripts. Ribosome RNA (rRNA) serves as a loading control. Mean values of band intensity in the northern blots quantified by ImageJ are shown below each blot. Note that the presence of CHV4 genomic RNA was confirmed in fungal strain C18 doubly infected MyRV2 and CHV4.

Figure 5

Enhanced infection stability of MyRV2 by transgenic expression of CHV4-C18 p24. (A,C) Detection of MyRV2 infection by RT-PCR in C18 transformants with the CHV4 p24 coding domain (C18p24) or the empty vector (C18emp). Virus infection in three transfectant subisolates from 5 independent transformants (lines 1 to 5) was monitored until 10th subcultures by colony RT-PCR. RT-PCR was carried out as described by Aulia et al. [28] using the primers CHV4-287_F and CHV4-853_R. Amplified fragments were analyzed in 1.2% agarose gel electrophoresis. (B,D) MyRV2 Infectivity after successive fungal subculture in C18p24 transformants (B) and transformants with the empty vector (C18emp) (D).

Figure 6

Transgenic expression of CHV4-C18 p24 in recipient C. parasitica strains facilitates horizontal transmission of MyRV2 via anastomosis. Non-transgenic donor isolates containing MyRV2 (left side of panel A) were co-cultured on PDA with CHV4-C18 p24 transformants (C18p24) as recipients (right side of panel A), or with virus-free (C18-VF) and CHV4-C18-infected (C18/CHV4) recipients as controls. Following anastomosis, subcultures were obtained from the recipient side and tested for MyRV2 infection by conventional dsRNA extraction and agarose gel electrophoresis (panel B).