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 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.

Identification of a Novel Hypovirulence-Inducing Hypovirus From Alternaria alternata

“…RNA secondary structure prediction showed the presence of multiple stem loops in the 5′-UTRs of AaHV1 and WhIV14, but the RNA conformations were different between those two viruses (Supplementary Figure S3B). Recently, the 5′-UTRs of hypoviruses (CHV1, CHV2, and CHV3) have been demonstrated to have an internal ribosomal entry site (IRES) function (Chiba et al, 2018), therefore it is of interest investigating whether the 5′-UTRs of AaHV1 and other hypo-like viruses, including WhIV14, have similar IRES activity.…”

Dicer functions transcriptionally and posttranscriptionally in a multilayer antiviral defense

“…For the dcl2 complementation experiment, the DNA fragment corresponding to the C. parasitica dcl2 ORF was inserted into the HpaI site of pCPXNeo (14) using the in-fusion cloning system (Clontech). Site-directed mutagenesis of dcl2 was carried out using two PCR steps, as described previously (51).…”

Diverse Partitiviruses From the Phytopathogenic Fungus, Rosellinia necatrix

“…The fungus is an attractive host because it is biologically tractable, genetically manipulatable, and can support many homologous and heterologous fungal viruses (Eusebio-Cope et al, 2015 ). In addition, a number of biological resources and molecular tools are available for the fungus (Nuss, 2005 ; Eusebio-Cope et al, 2015 ; Chiba et al, 2018 ). Therefore, the use of C. parasitica as a viral host has facilitated the study of viral symptom induction via host antiviral RNA silencing to and counter defense of both homologous and heterologous fungal viruses (Faruk et al, 2008 ; Andika et al, 2017 , 2019 ).…”