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

Human cytomegalovirus protein pUL36: A dual cell death pathway inhibitor.

Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of intrinsic, innate, and adaptive viral immune evasion. Here, we employed multiplexed tandem mass tag-based proteomics to characterize host proteins targeted for degradation late during HCMV infection. This approach revealed that mixed lineage kinase domain-like protein (MLKL), a key terminal mediator of cellular necroptosis, was rapidly and persistently degraded by the minimally passaged HCMV strain Merlin but not the extensively passaged strain AD169. The strain Merlin viral inhibitor of apoptosis pUL36 was necessary and sufficient both to degrade MLKL and to inhibit necroptosis. Furthermore, mutation of pUL36 Cys131 abrogated MLKL degradation and restored necroptosis. As the same residue is also required for pUL36-mediated inhibition of apoptosis by preventing proteolytic activation of procaspase-8, we define pUL36 as a multifunctional inhibitor of both apoptotic and necroptotic cell death

Human Cytomegalovirus Long Non-coding RNA1.2 Suppresses Extracellular Release of the Pro-inflammatory Cytokine IL-6 by Blocking NF-κB Activation.

Long non-coding RNAs (lncRNAs) are transcripts of >200 nucleotides that are not translated into functional proteins. Cellular lncRNAs have been shown to act as regulators by interacting with target nucleic acids or proteins and modulating their activities. We investigated the role of RNA1.2, which is one of four major lncRNAs expressed by human cytomegalovirus (HCMV), by comparing the properties of parental virus in vitro with those of deletion mutants lacking either most of the RNA1.2 gene or only the TATA element of the promoter. In comparison with parental virus, these mutants exhibited no growth defects and minimal differences in viral gene expression in human fibroblasts. In contrast, 76 cellular genes were consistently up- or down-regulated by the mutants at both the RNA and protein levels at 72 h after infection. Differential expression of the gene most highly upregulated by the mutants (Tumor protein p63-regulated gene 1-like protein; TPRG1L) was confirmed at both levels by RT-PCR and immunoblotting. Consistent with the known ability of TPRG1L to upregulate IL-6 expression via NF-κB stimulation, RNA1.2 mutant-infected fibroblasts were observed to upregulate IL-6 in addition to TPRG1L. Comparable surface expression of TNF receptors and responsiveness to TNF-α in cells infected by the parental and mutant viruses indicated that activation of signaling by TNF-α is not involved in upregulation of IL-6 by the mutants. In contrast, inhibition of NF-κB activity and knockdown of TPRG1L expression reduced the extracellular release of IL-6 by RNA1.2 mutant-infected cells, thus demonstrating that upregulation of TPRG1L activates NF-κB. The levels of MCP-1 and CXCL1 transcripts were also increased in RNA1.2 mutant-infected cells, further demonstrating the presence of active NF-κB signaling. These results suggest that RNA1.2 plays a role in manipulating intrinsic NF-κB-dependent cytokine and chemokine release during HCMV infection, thereby impacting downstream immune responses

Stability of Unicortical versus Bicortical Metacarpal Fracture Internal Fixation Trial (SUBMIT): study protocol for a randomized controlled trial

BACKGROUND: Metacarpal fractures are common, accounting for 40 % of all hand injuries. The use of plates for the fixation of these fractures allows early aggressive hand therapy post-operatively, reducing post-operative stiffness. Traditionally, bicortical fixation is the standard practice, where both dorsal and palmar cortices of the metacarpal are drilled through, with screws engaging both cortices. Recent biomechanical studies have shown that unicortical fixation, where only the near cortex is drilled and engaged by the screw, results in no difference in stiffness, load to failure or failure mechanism, when compared with bicortical fixation. This trial aims to compare fracture union, complication rate and functional outcomes between unicortical and bicortical fixation for adults with displaced metacarpal fractures. METHODS/DESIGN: All adults with displaced diaphyseal metacarpal fracture requiring plate fixation are potentially eligible to take part in this study. A total of 315 consenting patients will be randomly allocated to either unicortical or bicortical plate and screw fixation. The surgery will be performed in specialist hand trauma units across the UK. Data regarding fracture healing, hand function, quality of life, and complications will be collected at 2 weeks, 6 weeks and 6 months following surgery. DISCUSSION: This pragmatic, prospective, multi-centre, randomized controlled trial is expected to deliver results in 2018. TRIAL REGISTRATION: ISRCTN 18006607. Registered on 19 Nov 2015

Human cytomegalovirus interactome analysis identifies degradation hubs, domain associations and viral protein functions

Human cytomegalovirus (HCMV) extensively modulates host cells, downregulating >900 human proteins during viral replication and degrading ≥133 proteins shortly after infection. The mechanism of degradation of most host proteins remains unresolved, and the functions of many viral proteins are incompletely characterised. We performed a mass spectrometry-based interactome analysis of 169 tagged, stably-expressed canonical strain Merlin HCMV proteins, and two non-canonical HCMV proteins, in infected cells. This identified a network of >3,400 virus-host and >150 virus-virus protein interactions, providing insights into functions for multiple viral genes. Domain analysis predicted binding of the viral UL25 protein to SH3 domains of NCK Adaptor Protein-1. Viral interacting proteins were identified for 31/133 degraded host targets. Finally, the uncharacterised, non-canonical ORFL147C protein was found to interact with elements of the mRNA splicing machinery, and a mutational study suggested its importance in viral replication. The interactome data will be important for future studies of herpesvirus infection

Human cytomegalovirus protein RL1 degrades the antiviral factor SLFN11 via recruitment of the CRL4 E3 ubiquitin ligase complex.

Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of viral immune evasion, targeting intrinsic, innate, and adaptive immunity. We have employed two orthogonal multiplexed tandem mass tag-based proteomic screens to identify host proteins down-regulated by viral factors expressed during the latest phases of viral infection. This approach revealed that the HIV-1 restriction factor Schlafen-11 (SLFN11) was degraded by the poorly characterized, late-expressed HCMV protein RL1, via recruitment of the Cullin4-RING E3 Ubiquitin Ligase (CRL4) complex. SLFN11 potently restricted HCMV infection, inhibiting the formation and spread of viral plaques. Overall, we show that a restriction factor previously thought only to inhibit RNA viruses additionally restricts HCMV. We define the mechanism of viral antagonism and also describe an important resource for revealing additional molecules of importance in antiviral innate immunity and viral immune evasion

Climate Smart Disaster Risk Management

DfI

Human cytomegalovirus RNA2.7 is required for upregulating multiple cellular genes to promote cell motility and viral spread late in lytic infection

Long non-coding RNAs are frequently associated with broad modulation of gene expression and thus provide the cell with the ability to synchronize entire metabolic processes. We used transcriptomic approaches to investigate whether the most abundant human cytomegalovirus-encoded lncRNA, RNA2.7, has this characteristic. By comparing cells infected with wild-type virus (WT) with cells infected with RNA2.7 deletion mutants, RNA2.7 was implicated in regulating a large number of cellular genes late in lytic infection. Pathway analysis indicated that >100 of these genes are associated with promoting cell movement, and the ten most highly regulated of these were validated in further experiments. Morphological analysis and live cell tracking of WT- and RNA2.7 mutant-infected cells indicated that RNA2.7 is involved in promoting the movement and detachment of infected cells late in infection, and plaque assays using sparse cell monolayers indicated that RNA2.7 is also involved in promoting cell-to-cell spread of virus. Consistent with the observation that upregulated mRNAs are relatively A+U-rich, which is a trait associated with transcript instability, and that they are also enriched in motifs associated with mRNA instability, transcriptional inhibition experiments on WT- and RNA2.7 mutant-infected cells showed that four upregulated transcripts were longer-lived in the presence of RNA2.7. These findings demonstrate that RNA2.7 is required for promoting cell movement and viral spread late in infection and suggest that this may be due to general stabilization of A+U-rich transcripts