Identification and Characterization of Cyprinid Herpesvirus-3 (CyHV-3) Encoded MicroRNAs.

Owen H Donohoe,Keith Way,David M Stone,Kathy Henshilwood,Roya Hakimjavadi,Dermot Walls

doi:10.1371/journal.pone.0125434

Owen H Donohoe, Keith Way + Show 4 more

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https://doi.org/10.1371/journal.pone.0125434

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Abstract

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in post-transcriptional gene regulation. Some viruses encode their own miRNAs and these are increasingly being recognized as important modulators of viral and host gene expression. Cyprinid herpesvirus 3 (CyHV-3) is a highly pathogenic agent that causes acute mass mortalities in carp (Cyprinus carpio carpio) and koi (Cyprinus carpio koi) worldwide. Here, bioinformatic analyses of the CyHV-3 genome suggested the presence of non-conserved precursor miRNA (pre-miRNA) genes. Deep sequencing of small RNA fractions prepared from in vitro CyHV-3 infections led to the identification of potential miRNAs and miRNA–offset RNAs (moRNAs) derived from some bioinformatically predicted pre-miRNAs. DNA microarray hybridization analysis, Northern blotting and stem-loop RT-qPCR were then used to definitively confirm that CyHV-3 expresses two pre-miRNAs during infection in vitro. The evidence also suggested the presence of an additional four high-probability and two putative viral pre-miRNAs. MiRNAs from the two confirmed pre-miRNAs were also detected in gill tissue from CyHV-3-infected carp. We also present evidence that one confirmed miRNA can regulate the expression of a putative CyHV-3-encoded dUTPase. Candidate homologues of some CyHV-3 pre-miRNAs were identified in CyHV-1 and CyHV-2. This is the first report of miRNA and moRNA genes encoded by members of the Alloherpesviridae family, a group distantly related to the Herpesviridae family. The discovery of these novel CyHV-3 genes may help further our understanding of the biology of this economically important virus and their encoded miRNAs may have potential as biomarkers for the diagnosis of latent CyHV-3.

Highlights

MicroRNAs are a class of small non-coding RNAs involved in the regulation of gene expression in animals and plants [1,2,3,4]
Six other viral genomes known to collectively encode 94 pre-miRNAs were analysed so as to establish a set of relevant minimum cut-off values to be used for filtering out predictions that were less likely to represent genuine Cyprinid herpesvirus 3 (CyHV-3) pre-miRNAs
It is highly likely that the majority of those remaining predictions did not represent genuine pre-miRNAs, this strategy proved to be of use in identifying viral genomes with pre-miRNA coding capacity

Summary

Introduction

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in the regulation of gene expression in animals and plants [1,2,3,4]. They are ~22 nucleotides (nt) in length and are typically encoded within intronic or intergenic regions of genomes [5,6,7]. FC1193 and FC1202; KW), the Programme for Research in Third Level Institutions (PRTLI) (http:// www.hea.ie/en/prtli,official; RH, DW) Cycle 5 [The Bioanalysis and Therapeutics (BioAT) programme at DCU]. The PRTLI is co-funded through the European Regional Development Fund (ERDF), part of the European Union Structural Funds Programme 2007– 2013 (http://eustructuralfunds.gov.ie). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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