Validation and functional analysis of Ovine Herpesvirus 2-encoded microRNAs
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.