First Evidence for Internal Ribosomal Entry Sites in Diverse Fungal Virus Genomes
First Evidence for Internal Ribosomal Entry Sites in Diverse Fungal Virus Genomes
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DOI：
10.1128/mbio.02350-17
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发表时间：
2018-03-01
期刊：
影响因子：
6.4
通讯作者：
Suzuki, Nobuhiro
中科院分区：
文献类型：
--
作者：
Chiba, Sotaro;Jamal, Atif;Suzuki, Nobuhiro
与动物和植物中公认的内部核糖体进入位点（IRES）介导的翻译起始相反，在真菌病毒或细胞RNA中没有建立IRES。为了鉴定真菌病毒中的IRES元件，我们在栗疫病菌（一种用于病毒-宿主相互作用的丝状真菌）中开发了基于内切酶的双报告基因检测系统。双顺反子构建体需要密码子优化的海肾和萤火虫荧光素酶（分别为ORluc和OFluc）基因，在它们之间可以插入潜在的IRES序列。在该系统中，ORluc用作内部控制，而OFluc代表IRES活性。分析了不同正义单链RNA和双链RNA（dsRNA）病毒基因组的5'非翻译区（UTR）中的病毒序列。结果表明，对栗疫病菌低病毒1（CHV 1）和CHV 2的IRES活性相对较高，对CHV 3的活性微弱但可测量。CHV 3的弱IRES信号可以通过其单顺反子性质来解释，不同于CHV 1和CHV 2的双顺反子性质。这将允许这三种低病毒具有每个病毒mRNA分子的复制相关蛋白的相似翻译速率。证实了CHV 1的24个5 '-近端密码子以及5' UTR对于IRES功能的重要性。此外，维多利亚病毒和黄病毒检测IRES阳性，而呼肠孤病毒、分离病毒和四重病毒显示与阴性对照相似的Fluc活性。总的来说，这项研究代表了第一次开发的IRES鉴定系统在丝状真菌的基础上的密码子优化的双荧光素酶检测和IRES在丝状fungi.IMPORTANCE帽独立的证据，内部核糖体进入位点（IRES）介导的翻译起始经常被用于病毒的mRNA和罕见的细胞mRNA在动物和植物。然而，在真菌病毒RNA或丝状真菌的细胞RNA中尚未建立IRES。在这里，我们报告的双荧光素酶检测系统的发展和测量的IRES活动的真菌RNA病毒在模型丝状真菌宿主，栗疫病菌寄生。鉴定为IRES阳性的病毒包括次病毒（正义RNA病毒，扩展的小核糖核酸病毒超组的成员）、全病毒（非节段dsRNA病毒）和金病毒（四节段dsRNA病毒）。在呼肠孤病毒（11节段dsRNA病毒）、四节段dsRNA病毒（四节段dsRNA病毒）或二节段dsRNA病毒（二节段dsRNA病毒）的5'非翻译区中未观察到IRES活性。这项研究提供了IRES活动在丝状真菌中的不同RNA病毒的第一个证据，是确定反式作用的宿主因子和顺式调节病毒RNA元件的第一步。
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 mycoreo-viruses, 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.