Systems virology and human cytomegalovirus: Using high throughput approaches to identify novel host-virus interactions during lytic infection

Human Cytomegalovirus (HCMV) is a highly prevalent herpesvirus, persistently infecting between 30% and 100% of the population,depending on socio-economic status [1]. HCMV remains an important clinical pathogen accounting for more than 60% ofcomplications associated with solid organ transplant patients [2-4]. It is also the leading cause of infectious congenital birth defectsand has been linked to chronic inflammation and immune aging [5-7]. There is currently no effective vaccine and HCMV antiviralshave significant side effects. As current antivirals target viral genes, the virus can develop resistance, reducing drug efficacy.There is therefore an urgent need for new antiviral agents that are effective against HCMV, have better toxicity profiles and areless vulnerable to the emergence of resistant strains. Targeting of host factors that are critical to virus replication is a potentialstrategy for the development of novel antivirals that circumvent the development of viral resistance. Systematic high throughputapproaches provide powerful methods for the identification of novel host-virus interactions. As well as contributing to our basicunderstanding of virus and cell biology, such studies provide potential targets for the development of novel antiviral agents.High-throughput studies, such as RNA sequencing, proteomics, and RNA interference screens, are useful tools to identifyHCMV-induced global changes in host mRNA and protein expression levels and host factors important for virus replication. Here, wesummarise new findings on HCMV lytic infection from high-throughput studies since 2014 and how screening approaches haveevolved

The functions of herpesvirus-encoded microRNAs

Bioinformatic and direct cloning approaches have led to the identification of over 100 novel miRNAs expressed in DNA viruses, although the function of the majority of these small regulatory RNA molecules is unclear. Recently, a number of reports have now identified potential targets of viral miRNAs, including cellular and viral genes as well as an ortholog of an important immune-regulatory cellular miRNA. In this review, we will cover the identification and characterization of miRNAs expressed in the herpesvirus family and discuss the potential significance of their role in viral infection

Discovering antiviral restriction factors and pathways using genetic screens

Research in the Hughes lab is supported by a grant from the Academy of Medical Sciences (SFB003/1028), a grant from Tenovus Scotland (T20/63), and The Wellcome Trust Institutional Strategic Support Fund (ISSF). Research in the Gray lab is supported Medical Research Council (MR/N001796/1) and the Biotechnology and Biological Sciences Research Council (BBS/E/D/20002172). C. E. J. is supported by a University of St Andrews Ph.D. scholarship.Viral infections activate the powerful interferon (IFN) response that induces the expression of several hundred IFN stimulated genes (ISGs). The principal role of this extensive response is to create an unfavourable environment for virus replication and to limit spread; however, untangling the biological consequences of this large response is complicated. In addition to a seemingly high degree of redundancy, several ISGs are usually required in combination to limit infection as individual ISGs often have low to moderate antiviral activity. Furthermore, what ISG or combination of ISGs are antiviral for a given virus is usually not known. For these reasons, and that the function(s) of many ISGs remains unexplored, genome-wide approaches are well placed to investigate what aspects of this response results in an appropriate, virus-specific phenotype. This review discusses the advances screening approaches have provided for the study of host defence mechanisms, including CRISPR/Cas9, ISG expression libraries and RNAi technologies.Publisher PDFPeer reviewe

A Human Cytomegalovirus-Encoded microRNA Regulates Expression of Multiple Viral Genes Involved in Replication

Although multiple studies have documented the expression of over 70 novel virus-encoded microRNAs (miRNAs), the targets and functions of most of these regulatory RNA species are unknown. In this study a comparative bioinformatics approach was employed to identify potential human cytomegalovirus (HCMV) mRNA targets of the virus-encoded miRNA miR-UL112-1. Bioinformatics analysis of the known HCMV mRNA 3′ untranslated regions (UTRs) revealed 14 potential viral transcripts that were predicted to contain functional target sites for miR-UL112-1. The potential target sites were screened using luciferase reporters that contain the HCMV 3′UTRs in co-transfection assays with miR-UL112-1. Three of the 14 HCMV miRNA targets were validated, including the major immediate early gene encoding IE72 (UL123, IE1), UL112/113, and UL120/121. Further analysis of IE72 regulation by miR-UL112-1 with clones encoding the complete major immediate early region revealed that the IE72 3′UTR target site is necessary and sufficient to direct miR-UL112-1-specific inhibition of expression in transfected cells. In addition, miR-UL112-1 regulation is mediated through translational inhibition rather than RNA degradation. Premature expression of miR-UL112-1 during HCMV infection resulted in a significant decrease in genomic viral DNA levels, suggesting a functional role for miR-UL112-1 in regulating the expression of genes involved in viral replication. This study demonstrates the ability of a viral miRNA to regulate multiple viral genes

A Viral microRNA Down-Regulates Multiple Cell Cycle Genes through mRNA 5 ' UTRs

Global gene expression data combined with bioinformatic analysis provides strong evidence that mammalian miRNAs mediate repression of gene expression primarily through binding sites within the 3′ untranslated region (UTR). Using RNA induced silencing complex immunoprecipitation (RISC-IP) techniques we have identified multiple cellular targets for a human cytomegalovirus (HCMV) miRNA, miR-US25-1. Strikingly, this miRNA binds target sites primarily within 5′UTRs, mediating significant reduction in gene expression. Intriguingly, many of the genes targeted by miR-US25-1 are associated with cell cycle control, including cyclin E2, BRCC3, EID1, MAPRE2, and CD147, suggesting that miR-US25-1 is targeting genes within a related pathway. Deletion of miR-US25-1 from HCMV results in over expression of cyclin E2 in the context of viral infection. Our studies demonstrate that a viral miRNA mediates translational repression of multiple cellular genes by targeting mRNA 5′UTRs

Competition for Antigen at the Level of the APC Is a Major Determinant of Immunodominance during Memory Inflation in Murine Cytomegalovirus Infection

The unique ability of CMV to drive the expansion of virus-specific T cell populations during the course of a lifelong, persistent infection has generated interest in the virus as a potential vaccine strategy. When designing CMV-based vaccine vectors to direct immune responses against HIV or tumor Ags, it becomes important to understand how and why certain CMV-specific populations are chosen to inflate over time. To investigate this, we designed recombinant murine CMVs (MCMVs) encoding a SIINFEKL-enhanced GFP fusion protein under the control of endogenous immediate early promoters. When mice were infected with these viruses, T cells specific for the SIINFEKL epitope inflated and profoundly dominated T cells specific for nonrecombinant (i.e., MCMV-derived) Ags. Moreover, when the virus encoded SIINFEKL, T cells specific for nonrecombinant Ags displayed a phenotype indicative of less frequent exposure to Ag. The immunodominance of SIINFEKL-specific T cells could not be altered by decreasing the number of SIINFEKL-specific cells available to respond, or by increasing the number of cells specific for endogenous MCMV Ags. In contrast, coinfection with viruses expressing and lacking SIINFEKL enabled coinflation of T cells specific for both SIINFEKL and nonrecombinant Ags. Because coinfection allows presentation of SIINFEKL and MCMV-derived Ags by different cells within the same animal, these data reveal that competition for, or availability of, Ag at the level of the APC determines the composition of the inflationary response to MCMV. SIINFEKL\u27s strong affinity for H-2K(b), as well as its early and abundant expression, may provide this epitope\u27s competitive advantage

Cytomegalovirus MicroRNA Expression Is Tissue Specific and Is Associated with Persistence

MicroRNAs (miRNAs) are a class of small noncoding RNAs involved in posttranscriptional regulation. miRNAs are utilized in organisms ranging from plants to higher mammals, and data have shown that DNA viruses also use this method for host and viral gene regulation. Here, we report the sequencing of the small RNAs in rat cytomegalovirus (RCMV)-infected fibroblasts and persistently infected salivary glands. We identified 24 unique miRNAs that mapped to hairpin structures found within the viral genome. While most miRNAs were detected in both samples, four were detected exclusively in the infected fibroblasts and two were specific for the infected salivary glands. The RCMV miRNAs are distributed across the viral genome on both the positive and negative strands, with clusters of miRNAs at a number of locations, including near viral genes r1 and r111. The RCMV miRNAs have a genomic positional orientation similar to that of the miRNAs described for mouse cytomegalovirus, but they do not share any substantial sequence conservation. Similar to other reported miRNAs, the RCMV miRNAs had considerable variation at their 3′ and 5′ ends. Interestingly, we found a number of specific examples of differential isoform usage between the fibroblast and salivary gland samples. We determined by real-time PCR that expression of the RCMV miRNA miR-r111.1-2 is highly expressed in the salivary glands and that miR-R87-1 is expressed in most tissues during the acute infection phase. Our study identified the miRNAs expressed by RCMV in vitro and in vivo and demonstrated that expression is tissue specific and associated with a stage of viral infection

Human cytomegalovirus evades ZAP detection by suppressing CpG dinucleotides in the major immediate early 1 gene

The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG dinucleotide frequencies. Artificially increasing dinucleotide frequencies results in substantial attenuation of virus replication, suggesting that these compositional changes may facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct binding and possibly downstream targeting for degradation. Using an arrayed interferon stimulated gene expression library screen, we identified ZAPS, and its associated factor TRIM25, as inhibitors of human cytomegalovirus (HCMV) replication. Exogenous expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of CpG representation with specific suppression of CpG motifs within the IE1 major immediate early transcript which is absent in subsequently expressed genes. We demonstrated that suppression of CpG dinucleotides in the IE1 gene allows evasion of inhibitory effects of ZAP. We show that acute virus replication is mutually exclusive with high levels of cellular ZAP, potentially explaining the higher levels of CpG in viral genes expressed subsequent to IE1 due to the loss of pressure from ZAP in infected cells. Finally, we show that TRIM25 regulates alternative splicing between the ZAP short and long isoforms during HCMV infection and interferon induction, with knockdown of TRIM25 resulting in decreased ZAPS and corresponding increased ZAPL expression. These results demonstrate for the first time that ZAP is a potent host restriction factor against large DNA viruses and that HCMV evades ZAP detection through suppression of CpG dinucleotides within the major immediate early 1 transcript. Furthermore, TRIM25 is required for efficient upregulation of the interferon inducible short isoform of ZAP through regulation of alternative splicing

Ovine herpesvirus-2 encoded microRNAs target virus genes involved in virus latency

Herpesviruses encode miRNAs that target both virus and host genes; however their role in herpesvirus biology is poorly understood. We previously identified eight miRNAs encoded by OvHV-2; the causative agent of malignant catarrhal fever (MCF) and have now investigated the role of these miRNAs in regulating expression of OvHV-2 genes that play important roles in virus biology. ORF 20 (cell cycle inhibition), ORF 50 (reactivation) and ORF 73 (latency maintenance) each contain predicted targets for several OvHV-2 miRNAs. Co-transfection of miRNA mimics with luciferase reporter constructs containing the predicted targets showed the 5’ UTRs of ORF 20 and ORF 73 contain functional targets for ovhv-miR-2 and ovhv2-miR-8 respectively, and the 3’UTR of ORF 50 contains a functional target for ovhv2-miR-5. Transfection of BJ1035 cells (an OvHV-2 infected bovine T cell line) with the relevant miRNA mimic resulted in a significant decrease in ORF 50 and a smaller but non-significant decrease in ORF 20. However, we were unable to demonstrate a decrease in ORF 73. MCF is a disease of dysregulated lymphocyte proliferation, miRNA inhibition of ORF 20 expression may play a role in this aberrant lymphocyte proliferation. The proteins encoded by ORFs 50 and 73 play opposing roles in latency, it has been hypothesized that miRNA-induced inhibition of virus genes acts to ensure that fluctuations in virus mRNA levels do not result in reactivation in conditions that are unfavourable for viral replication, our data would support this hypothesis