Regulation of Ov2 by virus encoded microRNAs

Katie Nightingale,John Hopkins,Inga Dry,Robert Dalziel

doi:10.1007/s11259-019-09749-9

Katie Nightingale, John Hopkins + Show 2 more

Open Access

https://doi.org/10.1007/s11259-019-09749-9

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Abstract

Herpesviruses encode miRNAs that target both virus and host genes; however their role in herpesvirus biology is still poorly understood. We previously identified thirty five miRNAs encoded by OvHV-2; the causative agent of malignant catarrhal fever (MCF) and are investigating the role of these miRNAs in regulating expression of OvHV-2 genes that play important roles in virus biology. Analysis, using RNAHybrid predicted that two OvHV-2 encoded miRNAs, ovhv2-miR-17-10 and ovhv2-miR-61-1, target transcripts coding for the OvHV-2 bZIP protein Ov2. In other herpesvirus bZIP proteins are known to play important roles in lytic virus replication. Here we show by Flow cytometry and western blotting that ovhv2-miR-17-10 and ovhv2-miR-61-1, reduce the expression of Ov2 protein. The predicted target sites for both miRNAs within the Ov2 gene were disrupted whilst retaining the Ov2 coding sequence. Mutation of the ovhv2-miR-61-1 target sequence restored Ov2 protein expression levels to control levels confirming the identity of its target site. However, it was not possible to determine the binding site of ovhv2-miR-17-10 possibly due to potential G:U pairing introduced during the mutation process. The targeting of Ov2 by two virus-encoded miRNAs suggests an important regulatory role for Ov2 in OvHV-2 replication or reactivation.

Highlights

Malignant catarrhal fever (MCF) is a usually fatal disease of cattle, deer, bison and other ruminants caused by viruses in the genus Macavirus of the subfamily Gammaherpesvirinae (McGeoch et al 2006)
As there were potential off target effects of the ovhv2-miR61-1 on EGFP we investigated the effects of the OvHv-2 miRNA mimics to regulate expression of an untagged version of Ov2, by western blot analysis using an Ov2 specific antibody
Evidence is accumulating from a number of herpesvirus, including HCMVand Kaposi’s sarcoma-associated herpesvirus (KSHV), that virally-encoded miRNAs may function like a rheostat, in fine-tuning the mechanism of reactivation by targeting viral transactivators (Grey et al 2007; Bellare and Ganem 2009; Riaz et al 2014)

Summary

Introduction

Malignant catarrhal fever (MCF) is a usually fatal disease of cattle, deer, bison and other ruminants caused by viruses in the genus Macavirus of the subfamily Gammaherpesvirinae (McGeoch et al 2006). In the case of both the SA and WA- forms of MCF, reactivation of the virus from latency results in the production of infectious virions that can be transmitted to susceptible species, such as cattle, bison, or deer found in close proximity to asymptomatically shedding carrier animals. Herpesvirus proteins with bZIP domains include BZLF1 of Epstein Barr Virus (EBV) (Murata 2014), and the K8 protein of Kaposi’s sarcoma-associated herpesvirus (KSHV) (Lefort and Flamand 2009). BZLF and K8 play important roles in the lifecycles of EBV and KSHV by modulating the activity of RTA (Liao et al 2003; Murata 2014)

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