Validation and functional analysis of Ovine Herpesvirus 2-encoded microRNAs
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Nightingale2016.docx (13.41Mb)
Date
02/07/2016Author
Nightingale, Katie
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Abstract
Ovine herpesvirus 2 (OvHV-2) is a gammaherpesvirus of domestic sheep and causes the
lymphoproliferative disease malignant catarrhal fever (MCF) in susceptible ruminants,
including cattle. Sheep are latently infected but do not develop disease. MCF is characterised
by proliferation of non-antigen specific cytotoxic large granular lymphocytes which leads to
necrosis of infiltrated tissues and death. The molecular basis underlying MCF pathogenesis is
poorly understood and it is unknown what controls the differences in the clinical outcome of
infection between sheep and cattle, two closely related species.
microRNAs (miRNAs) are short noncoding RNAs that post-transcriptionally regulate gene
expression through targeting of mRNA. A number of herpesviruses have been shown to
encode miRNAs that are capable of regulating of both viral and cellular gene expression which
can often have an effect on the pathogenesis of the virus. Following RNA seq analysis of an
OvHV-2-infected bovine T-cell line (BJ1035) forty-five miRNAs were predicted to be
encoded. Eight miRNAs were previously validated by northern blotting, and a further twenty-seven
were confirmed using two PCR methods described in this project.
It was hypothesised that these virus-encoded miRNAs may differentially target cellular genes
in sheep and MCF-susceptible species. Previous work using the technique CLASH
(Crosslinking Ligation and Sequencing of Hybrids) identified Delta-like 1 (DLL1), a ligand
for Notch signalling, as a potential target of ovhv2-miR-17-2. Initially, differential targeting
of DLL1 between sheep and cattle was hypothesised due to differences in the sequence and
number of binding sites for ovhv2-miR-17-2. The sheep DLL1 mRNA was shown to be
targeted however, due to incorrect annotation of the sheep genome, targeting of DLL1 is likely
in both sheep and cattle. One OvHV-2-encoded miRNA, ovhv2-miR-73-1, has partial
homology to a mammalian miRNA, miR-216a. Based on this homology it was predicted that
ovhv2-miR-73-1 may target Phosphatase and Tensin Homolog (PTEN) and Y Box Binding
Protein 1 (YB-1), as they are known targets of miR-216a. A GFP-reporter system was used to
demonstrate that despite having similar seed sequences, ovhv2-miR-73-1 does not target
PTEN or YB-1. Bioinformatic prediction was used to identify MHC class II genes as potential
targets of OvHV-2-encoded miRNAs. Two miRNAs, ovhv2-miR-17-25 and ovhv2-miR-17-9
were shown to target sheep MHC class II genes (DRA and DQB respectively) using a luciferase
reporter system. These miRNAs were not predicted to target the equivalent genes in cattle
indicating that these genes may be differentially regulated between sheep and cattle.
It was also shown that two OvHV-2-encoded miRNAs, ovhv2-miR-17-10 and ovhv2-miR-61-1, target the viral protein Ov2. Ov2 is predicted to contain a basic leucine zipper (bZIP) domain
and is therefore likely to be a transcription factor. Other closely related gammaherpesviruses
encode proteins that contain bZIP domains and these play major roles in the reactivation of
the virus from latency. Immunofluorescence and confocal microscopy was performed to
confirm the nuclear localisation of Ov2. RT-qPCRs were performed to investigate whether
Ov2 could regulate the expression of any cellular genes. Of the two genes investigated, one of
these, Jagged (JAG1), was downregulated in the presence of an Ov2-EGFPN1 construct
compared to a control plasmid. JAG1 is another ligand for Notch signalling indicating that the
virus may manipulate Notch signalling using multiple methods. Immunoprecipitation and
mass spectrometry analysis of an Ov2HA-pcDNA3.1+ construct was performed and a number
of potential interacting partners of Ov2 were identified.