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. 2018 Mar 20;9(2):e02350-17.
doi: 10.1128/mBio.02350-17.

First Evidence for Internal Ribosomal Entry Sites in Diverse Fungal Virus Genomes

Sotaro Chiba  1   2   3 , Atif Jamal  1   4 , Nobuhiro Suzuki  5
Affiliations

First Evidence for Internal Ribosomal Entry Sites in Diverse Fungal Virus Genomes

Sotaro Chiba et al. mBio. .

Abstract

In contrast to well-established internal ribosomal entry site (IRES)-mediated translational initiation in animals and plants, no IRESs were established in fungal viral or cellular RNAs. To identify IRES elements in mycoviruses, we developed a luciferase-based dual-reporter detection system in Cryphonectria parasitica, a model filamentous fungus for virus-host interactions. A bicistronic construct entails a codon-optimized Renilla and firefly luciferase (ORluc and OFluc, respectively) gene, between which potential IRES sequences can be inserted. In this system, ORluc serves as an internal control, while OFluc represents IRES activity. Virus sequences in the 5' untranslated regions (UTRs) of the genomes of diverse positive-sense single-stranded RNA and double-stranded RNA (dsRNA) viruses were analyzed. The results show relatively high IRES activities for Cryphonectria hypovirus 1 (CHV1) and CHV2 and faint but measurable activity for CHV3. The weak IRES signal of CHV3 may be explained by its monocistronic nature, differing from the bicistronic nature of CHV1 and CHV2. This would allow these three hypoviruses to have similar rates of translation of replication-associated protein per viral mRNA molecule. The importance of 24 5'-proximal codons of CHV1 as well as the 5' UTR for IRES function was confirmed. Furthermore, victoriviruses and chrysoviruses tested IRES positive, whereas mycoreoviruses, partitiviruses, and quadriviruses showed similar Fluc activities as the negative controls. Overall, this study represents the first development of an IRES identification system in filamentous fungi based on the codon-optimized dual-luciferase assay and provides evidence for IRESs in filamentous fungi.IMPORTANCE Cap-independent, internal ribosomal entry site (IRES)-mediated translational initiation is often used by virus mRNAs and infrequently by cellular mRNAs in animals and plants. However, no IRESs have been established in fungal virus RNAs or cellular RNAs in filamentous fungi. Here, we report the development of a dual-luciferase assay system and measurement of the IRES activities of fungal RNA viruses in a model filamentous fungal host, Cryphonectria parasitica Viruses identified as IRES positive include hypoviruses (positive-sense RNA viruses, members of the expanded Picornavirus supergroup), totiviruses (nonsegmented dsRNA viruses), and chrysoviruses (tetrasegmented dsRNA viruses). No IRES activities were observed in the 5' untranslated regions of mycoreoviruses (11-segmented dsRNA viruses), quadriviruses (tetrasegmented dsRNA viruses), or partitiviruses (bisegmented dsRNA viruses). This study provides the first evidence for IRES activities in diverse RNA viruses in filamentous fungi and is a first step toward identifying trans-acting host factors and cis-regulatory viral RNA elements.

Keywords: dsRNA virus; hypovirus; internal ribosome entry site; mycovirus; noncanonical translation.

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Figures

FIG 1
FIG 1
Development of the dual-luciferase (DL) reporter system in C. parasitica. (A) Construction of transformation vectors expressing luciferase genes. Original or codon-optimized Renilla and firefly luciferase genes (Rluc and Fluc or ORluc and OFluc, respectively) were cloned into the pCPXHY3 vector, and pCH-Rluc, pCH-Fluc, pCH-ORluc, and pCH-OFluc were obtained. The wild-type (WT) C. parasitica EP155 standard strain was transformed with these constructs and served as a negative control (dotted lines in C). (B) Schematic representation of the dual-luciferase reporter assay performed in this study. (C) Effectiveness of codon-optimized luciferase genes in C. parasitica. Luciferase activities were measured for transformants and a nontransformed WT strain as a reference. The raw relative luminescence units (RLU) as integrated values in 12 s are presented. (D) Confirmation of the dual-luciferase system in C. parasitica. Mycelia of WT or ORluc- or OFluc-expressing lines were homogenized. The OFluc-expressing line (OFluc, half diluted with PBS), a mixture of WT and OFluc-expressing lines (OFluc+WT), or a mixture of OFluc- and ORluc-expressing lines (OFluc+ORluc) was subjected to analysis. RLU were measured at three time points: 1, firefly luciferase measurement; 2, quenching step by addition of Renilla firefly buffer; and 3, Renilla luciferase measurement by addition of the substrate for Renilla luciferase, as shown in panel B.
FIG 2
FIG 2
Detection of hypovirus IRES elements. (A and B) Schematic representation of the IRES identification foundation construct, pCH-DLst3. ORluc and OFluc genes are tandemly inserted in the pCPXHY3 expression vector, under the control of the gpd-1 promoter and terminator (A). The ORluc gene is terminated with a stop codon triplet. A multiple cloning site (MCS) is created between the luciferase genes (B). (C and D) IRES detection by dual-luciferase (DL) reporter assays. C. parasitica hypovirus-originating sequences were analyzed. The 5′ untranslated region (UTR) and the adjacent 72 nt from the 5′-proximal ORF region of CHV1, CHV2, and CHV3 were cloned in pCH-DLst3 (C). The 72-nt sequences are in frame to the downstream OFluc gene. The antisense sequence of the CHV1 5′ UTR was used as a negative control (CHV1-as). The DL assay with 1 s of measurement was conducted, and the ratio of OFluc RLU to ORluc RLU (OFluc RLU standardized by ORluc RLU [F/R ratio]) was calculated and graphically shown (D). Open boxes, coding regions; black lines, plasmid sequences; orange lines, viral UTRs; light green line, antisense of CHV1 UTR; gray boxes, small cistrons (consisting of over 9 nt) on 5′ UTRs in three different frames.
FIG 3
FIG 3
Deletion analysis of CHV1 5′-proximal coding region. The DL assay with 1 s of measurement was conducted using C. parasitica transformed with pCH-DLst3 (empty) and its variant carrying the CHV1 5′ UTR (CHV1_wt) without or with deletions/substitution (CHV1_CR1, _CR2, _CR3, or _ΔAUG) in 72 nt from the first ORF. The F/R ratio was calculated as the percentage relative to pCH-DLst3-CHV1 (100%) and numerically presented (values in parentheses are standard deviations). Introduced mutations are schematically represented with a close view of the coding region. Black lines, retained sequence; red dashed lines, deleted regions; black and red dots, AUG start codon and its CCG substitutions, respectively. The expected RNA structure of the CHV1-EP713 5′ terminal region is adopted and depicted based on the work of Mu et al. (53) (see Fig. S3 for the predicted RNA structure of the CHV1 5′ UTR).
FIG 4
FIG 4
Detection of IRES elements carried by dsRNA mycoviruses. (A) Construction of transformation vectors carrying dsRNA viral cDNA sequences in pCH-DLst3. Lengths of viral 5′ UTRs (nucleotides) and detectable minicistrons over 9 nt are shown as in Fig. 2 (monotone). (B) Dual-luciferase assays. The DL assay was conducted using constructs shown in panel A at 1 s of measurement for both the luciferases. Ratios of OFluc RLU to ORluc RLU (F/R ratios) were calculated and graphically shown. An F/R ratio of less than 0.001 is indicated by an asterisk, representing no IRES activity.
FIG 5
FIG 5
Validation of detected IRESs. (A) Transient IRES assay in fungal protoplasts. In vitro-synthesized RNAs carrying DL with the CHV1, CHV2, CHV3, MyRV1, RnVV1, HvV145S, or RnQV1 sequence were introduced into C. parasitica cells by electroporation. The DL assay was then conducted at 3 h after electroporation with 12 s of measurement, together with the empty vector (pBS-DLst3) as a negative control. The F/R ratio was calculated, and results from three independent experiments were statistically analyzed as one set of data. (B) IRES activities of CHV1 UTR variants. As performed in panel A, IRES activities were evaluated in the RNA transfection-based transient assay for the constructs CHV1_wt, CHV1_CR1, and CHV1_CR3 together with the empty vector (pBS-DLst3) as a negative control.
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