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

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...

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Autores principales:	Sotaro Chiba, Atif Jamal, Nobuhiro Suzuki
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	dsRNA virus hypovirus internal ribosome entry site mycovirus noncanonical translation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/1d3d15631ccd4166998dc45770fd38a7
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spelling	oai:doaj.org-article:1d3d15631ccd4166998dc45770fd38a72021-11-15T15:53:26ZFirst Evidence for Internal Ribosomal Entry Sites in Diverse Fungal Virus Genomes10.1128/mBio.02350-172150-7511https://doaj.org/article/1d3d15631ccd4166998dc45770fd38a72018-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02350-17https://doaj.org/toc/2150-7511ABSTRACT 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.Sotaro ChibaAtif JamalNobuhiro SuzukiAmerican Society for MicrobiologyarticledsRNA virushypovirusinternal ribosome entry sitemycovirusnoncanonical translationMicrobiologyQR1-502ENmBio, Vol 9, Iss 2 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	dsRNA virus hypovirus internal ribosome entry site mycovirus noncanonical translation Microbiology QR1-502
spellingShingle	dsRNA virus hypovirus internal ribosome entry site mycovirus noncanonical translation Microbiology QR1-502 Sotaro Chiba Atif Jamal Nobuhiro Suzuki First Evidence for Internal Ribosomal Entry Sites in Diverse Fungal Virus Genomes
description	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.
format	article
author	Sotaro Chiba Atif Jamal Nobuhiro Suzuki
author_facet	Sotaro Chiba Atif Jamal Nobuhiro Suzuki
author_sort	Sotaro Chiba
title	First Evidence for Internal Ribosomal Entry Sites in Diverse Fungal Virus Genomes
title_short	First Evidence for Internal Ribosomal Entry Sites in Diverse Fungal Virus Genomes
title_full	First Evidence for Internal Ribosomal Entry Sites in Diverse Fungal Virus Genomes
title_fullStr	First Evidence for Internal Ribosomal Entry Sites in Diverse Fungal Virus Genomes
title_full_unstemmed	First Evidence for Internal Ribosomal Entry Sites in Diverse Fungal Virus Genomes
title_sort	first evidence for internal ribosomal entry sites in diverse fungal virus genomes
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/1d3d15631ccd4166998dc45770fd38a7
work_keys_str_mv	AT sotarochiba firstevidenceforinternalribosomalentrysitesindiversefungalvirusgenomes AT atifjamal firstevidenceforinternalribosomalentrysitesindiversefungalvirusgenomes AT nobuhirosuzuki firstevidenceforinternalribosomalentrysitesindiversefungalvirusgenomes
_version_	1718427259779416064

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

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