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1.  A Loss of Function Analysis of Host Factors Influencing Vaccinia virus Replication by RNA Interference 
PLoS ONE  2014;9(6):e98431.
Vaccinia virus (VACV) is a large, cytoplasmic, double-stranded DNA virus that requires complex interactions with host proteins in order to replicate. To explore these interactions a functional high throughput small interfering RNA (siRNA) screen targeting 6719 druggable cellular genes was undertaken to identify host factors (HF) influencing the replication and spread of an eGFP-tagged VACV. The experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellular proteins involved in cell-to-cell spread of VACV. The screen revealed 153 pro- and 149 anti-viral HFs that strongly influenced VACV replication. These HFs were investigated further by comparisons with transcriptional profiling data sets and HFs identified in RNAi screens of other viruses. In addition, functional and pathway analysis of the entire screen was carried out to highlight cellular mechanisms involved in VACV replication. This revealed, as anticipated, that many pro-viral HFs are involved in translation of mRNA and, unexpectedly, suggested that a range of proteins involved in cellular transcriptional processes and several DNA repair pathways possess anti-viral activity. Multiple components of the AMPK complex were found to act as pro-viral HFs, while several septins, a group of highly conserved GTP binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral VACV HFs. This screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. This advancement in our understanding of the VACV life cycle provides a reliable knowledge base for the improvement of poxvirus-based vaccine vectors and development of anti-viral theraputics.
PMCID: PMC4047015  PMID: 24901222
2.  Parallel classification and feature selection in microarray data using SPRINT 
The statistical language R is favoured by many biostatisticians for processing microarray data. In recent times, the quantity of data that can be obtained in experiments has risen significantly, making previously fast analyses time consuming or even not possible at all with the existing software infrastructure. High performance computing (HPC) systems offer a solution to these problems but at the expense of increased complexity for the end user. The Simple Parallel R Interface is a library for R that aims to reduce the complexity of using HPC systems by providing biostatisticians with drop-in parallelised replacements of existing R functions. In this paper we describe parallel implementations of two popular techniques: exploratory clustering analyses using the random forest classifier and feature selection through identification of differentially expressed genes using the rank product method.
PMCID: PMC4038771  PMID: 24883047
HPC; Genomics; Parallel programming
3.  Uterine Natural Killer cells regulate endometrial bleeding in women and are suppressed by the progesterone receptor modulator asoprisnil 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(5):10.4049/jimmunol.1300958.
Uterine NK cells (uNK) play a role in the regulation of placentation but their functions in non-pregnant endometrium are not understood. We have previously reported suppression of endometrial bleeding and alteration of spiral artery morphology in women exposed to asoprisnil, a progesterone receptor modulator. We now compare global endometrial gene expression in asoprisnil-treated versus control women, and we demonstrate a statistically significant reduction of genes in the IL-15 pathway, known to play a key role in uNK development and function. Suppression of IL-15 by asoprisnil was also observed at mRNA level (p<0.05), and immunostaining for NK cell marker CD56 revealed a striking reduction of uNK in asoprisnil-treated endometrium (p<0.001). IL-15 levels in normal endometrium are progesterone-responsive. Progesterone receptor (PR) positive stromal cells transcribe both IL-15 and IL-15RA. Thus, the response of stromal cells to progesterone will be to increase IL-15 trans-presentation to uNK, supporting their expansion and differentiation. In asoprisnil-treated endometrium, there is a marked down-regulation of stromal PR expression and virtual absence of uNK. These novel findings indicate that the IL-15 pathway provides a missing link in the complex interplay between endometrial stromal cells, uNK and spiral arteries affecting physiological and pathological endometrial bleeding.
PMCID: PMC3843142  PMID: 23913972
Endometrium; spiral arteries; NK cells; IL-15
4.  Viral Enhancer Mimicry of Host Innate-Immune Promoters 
PLoS Pathogens  2014;10(2):e1003804.
PMCID: PMC3916337  PMID: 24516378
5.  A Systematic Analysis of Host Factors Reveals a Med23-Interferon-λ Regulatory Axis against Herpes Simplex Virus Type 1 Replication 
PLoS Pathogens  2013;9(8):e1003514.
Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to identify HFs critical for disease progression and outcome.
Author Summary
Herpes simplex virus type 1 (HSV-1) infects the vast majority of the global population. Whilst most people experience the relatively mild symptoms of cold sores, some individuals suffer more serious diseases like viral meningitis and encephalitis. HSV-1 is also becoming more common as a cause of genital herpes, traditionally associated with HSV-2 infection. Co-infection with HSV-2 is a major contributor to HIV transmission, so a better understanding of HSV-1/HSV-2 disease has wide implications for global healthcare. After initial infection, all herpesviruses have the ability to remain dormant, and can awaken to cause a symptomatic infection at any stage. Whether the virus remains dormant or active is the result of a finely tuned balance between our immune system and evasion techniques developed by the virus. In this study we have found a new method by which the replication of the virus is counteracted. The cellular protein Med23 was found to actively induce an innate anti-viral immune response in the form of the Type III interferons (IFN-lambda), by binding IRF7, a key regulator of interferons, and modulating its activity. Interferon lambda is well known to be important in the control of Hepatitis C infection, and a genetic mutation correlating to an increase in interferon lambda levels is strongly linked to clearance of infection. Here we find the same association between this genetic mutation and the clinical severity of recurrent cases of HSV-1 infection (coldsores). These data identify a Med23-interferon lambda regulatory axis of innate immunity, show that interferon lambda plays a significant role in HSV-1 infection, and contribute to the expanding evidence for interferon lambda in disease control.
PMCID: PMC3738494  PMID: 23950709
6.  Deciphering the modulation of gene expression by type I and II interferons combining 4sU-tagging, translational arrest and in silico promoter analysis 
Nucleic Acids Research  2013;41(17):8107-8125.
Interferons (IFN) play a pivotal role in innate immunity, orchestrating a cell-intrinsic anti-pathogenic state and stimulating adaptive immune responses. The complex interplay between the primary response to IFNs and its modulation by positive and negative feedback loops is incompletely understood. Here, we implement the combination of high-resolution gene-expression profiling of nascent RNA with translational inhibition of secondary feedback by cycloheximide. Unexpectedly, this approach revealed a prominent role of negative feedback mechanisms during the immediate (≤60 min) IFNα response. In contrast, a more complex picture involving both negative and positive feedback loops was observed on IFNγ treatment. IFNγ-induced repression of genes associated with regulation of gene expression, cellular development, apoptosis and cell growth resulted from cycloheximide-resistant primary IFNγ signalling. In silico promoter analysis revealed significant overrepresentation of SP1/SP3-binding sites and/or GC-rich stretches. Although signal transducer and activator of transcription 1 (STAT1)-binding sites were not overrepresented, repression was lost in absence of STAT1. Interestingly, basal expression of the majority of these IFNγ-repressed genes was dependent on STAT1 in IFN-naïve fibroblasts. Finally, IFNγ-mediated repression was also found to be evident in primary murine macrophages. IFN-repressed genes include negative regulators of innate and stress response, and their decrease may thus aid the establishment of a signalling perceptive milieu.
PMCID: PMC3783172  PMID: 23832230
7.  A comprehensive machine-readable view of the mammalian cholesterol biosynthesis pathway 
Biochemical Pharmacology  2013;86(1):56-66.
Graphical abstract
We present the pathways leading to cholesterol, epoxy-cholesterol and oxysterol synthesis.
Cholesterol biosynthesis serves as a central metabolic hub for numerous biological processes in health and disease. A detailed, integrative single-view description of how the cholesterol pathway is structured and how it interacts with other pathway systems is lacking in the existing literature. Here we provide a systematic review of the existing literature and present a detailed pathway diagram that describes the cholesterol biosynthesis pathway (the mevalonate, the Kandutch-Russell and the Bloch pathway) and shunt pathway that leads to 24(S),25-epoxycholesterol synthesis. The diagram has been produced using the Systems Biology Graphical Notation (SBGN) and is available in the SBGN-ML format, a human readable and machine semantically parsable open community file format.
PMCID: PMC3912678  PMID: 23583456
Cholesterol; Sterol; SBGN; Metabolic network; Pathway map
8.  A model of flux regulation in the cholesterol biosynthesis pathway: Immune mediated graduated flux reduction versus statin-like led stepped flux reduction 
Biochimie  2013;95(3):613-621.
The cholesterol biosynthesis pathway has recently been shown to play an important role in the innate immune response to viral infection with host protection occurring through a coordinate down regulation of the enzymes catalysing each metabolic step. In contrast, statin based drugs, which form the principle pharmaceutical agents for decreasing the activity of this pathway, target a single enzyme. Here, we build an ordinary differential equation model of the cholesterol biosynthesis pathway in order to investigate how the two regulatory strategies impact upon the behaviour of the pathway. We employ a modest set of assumptions: that the pathway operates away from saturation, that each metabolite is involved in multiple cellular interactions and that mRNA levels reflect enzyme concentrations. Using data taken from primary bone marrow derived macrophage cells infected with murine cytomegalovirus or treated with IFNγ, we show that, under these assumptions, coordinate down-regulation of enzyme activity imparts a graduated reduction in flux along the pathway. In contrast, modelling a statin-like treatment that achieves the same degree of down-regulation in cholesterol production, we show that this delivers a step change in flux along the pathway. The graduated reduction mediated by physiological coordinate regulation of multiple enzymes supports a mechanism that allows a greater level of specificity, altering cholesterol levels with less impact upon interactions branching from the pathway, than pharmacological step reductions. We argue that coordinate regulation is likely to show a long-term evolutionary advantage over single enzyme regulation. Finally, the results from our models have implications for future pharmaceutical therapies intended to target cholesterol production with greater specificity and fewer off target effects, suggesting that this can be achieved by mimicking the coordinated down-regulation observed in immunological responses.
Graphical abstract
► We model the cholesterol biosynthesis pathway and its regulation. ► The innate immune response leads to a suppression of flux through the pathway. ► Statin inhibitors show a different mode of suppression to the immune response. ► Statin inhibitor suppression is less robust and less specific than immune suppression.
PMCID: PMC3585962  PMID: 22664637
Cholesterol; Systems biology; Regulation; Anti-viral; Statin
9.  The Transcription Factor STAT-1 Couples Macrophage Synthesis of 25-Hydroxycholesterol to the Interferon Antiviral Response 
Immunity  2013;38(1):106-118.
Recent studies suggest that the sterol metabolic network participates in the interferon (IFN) antiviral response. However, the molecular mechanisms linking IFN with the sterol network and the identity of sterol mediators remain unknown. Here we report a cellular antiviral role for macrophage production of 25-hydroxycholesterol (cholest-5-en-3β,25-diol, 25HC) as a component of the sterol metabolic network linked to the IFN response via Stat1. By utilizing quantitative metabolome profiling of all naturally occurring oxysterols upon infection or IFN-stimulation, we reveal 25HC as the only macrophage-synthesized and -secreted oxysterol. We show that 25HC can act at multiple levels as a potent paracrine inhibitor of viral infection for a broad range of viruses. We also demonstrate, using transcriptional regulatory-network analyses, genetic interventions and chromatin immunoprecipitation experiments that Stat1 directly coupled Ch25h regulation to IFN in macrophages. Our studies describe a physiological role for 25HC as a sterol-lipid effector of an innate immune pathway.
Graphical Abstract
► Macrophage PRR sensing of virus or IFN activation induce 25HC synthesis and secretion ► Stat1 rapidly binds and activates the promoter of cholesterol-25-hydroxylase (Ch25h) ► 25HC exerts multilevel antiviral function for a range of different viruses ► 25HC is an intrinsic paracrine and autocrine effector of the IFN antiviral response
PMCID: PMC3556782  PMID: 23273843
11.  Ablation of the Regulatory IE1 Protein of Murine Cytomegalovirus Alters In Vivo Pro-inflammatory TNF-alpha Production during Acute Infection 
PLoS Pathogens  2012;8(8):e1002901.
Little is known about the role of viral genes in modulating host cytokine responses. Here we report a new functional role of the viral encoded IE1 protein of the murine cytomegalovirus in sculpting the inflammatory response in an acute infection. In time course experiments of infected primary macrophages (MΦs) measuring cytokine production levels, genetic ablation of the immediate-early 1 (ie1) gene results in a significant increase in TNFα production. Intracellular staining for cytokine production and viral early gene expression shows that TNFα production is highly associated with the productively infected MΦ population of cells. The ie1- dependent phenotype of enhanced MΦ TNFα production occurs at both protein and RNA levels. Noticeably, we show in a series of in vivo infection experiments that in multiple organs the presence of ie1 potently inhibits the pro-inflammatory cytokine response. From these experiments, levels of TNFα, and to a lesser extent IFNβ, but not the anti-inflammatory cytokine IL10, are moderated in the presence of ie1. The ie1- mediated inhibition of TNFα production has a similar quantitative phenotype profile in infection of susceptible (BALB/c) and resistant (C57BL/6) mouse strains as well as in a severe immuno-ablative model of infection. In vitro experiments with infected macrophages reveal that deletion of ie1 results in increased sensitivity of viral replication to TNFα inhibition. However, in vivo infection studies show that genetic ablation of TNFα or TNFRp55 receptor is not sufficient to rescue the restricted replication phenotype of the ie1 mutant virus. These results provide, for the first time, evidence for a role of IE1 as a regulator of the pro-inflammatory response and demonstrate a specific pathogen gene capable of moderating the host production of TNFα in vivo.
Author Summary
The suppression of the production rather than the blockage of action of the potent inflammatory mediator TNFα is a particular hallmark of anti-TNFα mechanisms associated with microbial and parasitic infections. Whether this mode of counter-regulation is an important feature of infection by viruses is not clear. Also, it remains to be determined whether a specific pathogen gene in the context of an infection in vivo is capable of modulating levels of TNFα production. In this study we disclose a virus-mediated moderation of TNFα production, dependent on the ie1 gene of murine cytomegalovirus (MCMV). The ie1 gene product IE1 is a well-characterized nuclear protein capable of altering levels of host and viral gene expression although its biological role in the context of a natural infection is to date unknown. We provide evidence showing that ie1 is associated with a moderated pro-inflammatory cytokine response, in particular with TNFα production. Further, we show that the viral moderation of this cytokine is not only readily apparent in vitro but also in the natural host. The identification of a viral gene responsible for this mode of regulation in vivo may have therapeutic potential in the future in both anti-viral and anti-inflammatory strategies.
PMCID: PMC3431344  PMID: 22952450
12.  Digital clocks: simple Boolean models can quantitatively describe circadian systems 
The gene networks that comprise the circadian clock modulate biological function across a range of scales, from gene expression to performance and adaptive behaviour. The clock functions by generating endogenous rhythms that can be entrained to the external 24-h day–night cycle, enabling organisms to optimally time biochemical processes relative to dawn and dusk. In recent years, computational models based on differential equations have become useful tools for dissecting and quantifying the complex regulatory relationships underlying the clock's oscillatory dynamics. However, optimizing the large parameter sets characteristic of these models places intense demands on both computational and experimental resources, limiting the scope of in silico studies. Here, we develop an approach based on Boolean logic that dramatically reduces the parametrization, making the state and parameter spaces finite and tractable. We introduce efficient methods for fitting Boolean models to molecular data, successfully demonstrating their application to synthetic time courses generated by a number of established clock models, as well as experimental expression levels measured using luciferase imaging. Our results indicate that despite their relative simplicity, logic models can (i) simulate circadian oscillations with the correct, experimentally observed phase relationships among genes and (ii) flexibly entrain to light stimuli, reproducing the complex responses to variations in daylength generated by more detailed differential equation formulations. Our work also demonstrates that logic models have sufficient predictive power to identify optimal regulatory structures from experimental data. By presenting the first Boolean models of circadian circuits together with general techniques for their optimization, we hope to establish a new framework for the systematic modelling of more complex clocks, as well as other circuits with different qualitative dynamics. In particular, we anticipate that the ability of logic models to provide a computationally efficient representation of system behaviour could greatly facilitate the reverse-engineering of large-scale biochemical networks.
PMCID: PMC3405750  PMID: 22499125
systems biology; circadian gene networks; Boolean logic; photoperiodism; Arabidopsis thaliana
13.  Reversible Inhibition of Murine Cytomegalovirus Replication by Gamma Interferon (IFN-γ) in Primary Macrophages Involves a Primed Type I IFN-Signaling Subnetwork for Full Establishment of an Immediate-Early Antiviral State ▿ †  
Journal of Virology  2011;85(19):10286-10299.
Activated macrophages play a central role in controlling inflammatory responses to infection and are tightly regulated to rapidly mount responses to infectious challenge. Type I interferon (alpha/beta interferon [IFN-α/β]) and type II interferon (IFN-γ) play a crucial role in activating macrophages and subsequently restricting viral infections. Both types of IFNs signal through related but distinct signaling pathways, inducing a vast number of interferon-stimulated genes that are overlapping but distinguishable. The exact mechanism by which IFNs, particularly IFN-γ, inhibit DNA viruses such as cytomegalovirus (CMV) is still not fully understood. Here, we investigate the antiviral state developed in macrophages upon reversible inhibition of murine CMV by IFN-γ. On the basis of molecular profiling of the reversible inhibition, we identify a significant contribution of a restricted type I IFN subnetwork linked with IFN-γ activation. Genetic knockout of the type I-signaling pathway, in the context of IFN-γ stimulation, revealed an essential requirement for a primed type I-signaling process in developing a full refractory state in macrophages. A minimal transient induction of IFN-β upon macrophage activation with IFN-γ is also detectable. In dose and kinetic viral replication inhibition experiments with IFN-γ, the establishment of an antiviral effect is demonstrated to occur within the first hours of infection. We show that the inhibitory mechanisms at these very early times involve a blockade of the viral major immediate-early promoter activity. Altogether our results show that a primed type I IFN subnetwork contributes to an immediate-early antiviral state induced by type II IFN activation of macrophages, with a potential further amplification loop contributed by transient induction of IFN-β.
PMCID: PMC3196417  PMID: 21775459
14.  Viral Mediated Redirection of NEMO/IKKγ to Autophagosomes Curtails the Inflammatory Cascade 
PLoS Pathogens  2012;8(2):e1002517.
The early host response to viral infections involves transient activation of pattern recognition receptors leading to an induction of inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα). Subsequent activation of cytokine receptors in an autocrine and paracrine manner results in an inflammatory cascade. The precise mechanisms by which viruses avert an inflammatory cascade are incompletely understood. Nuclear factor (NF)-κB is a central regulator of the inflammatory signaling cascade that is controlled by inhibitor of NF-κB (IκB) proteins and the IκB kinase (IKK) complex. In this study we show that murine cytomegalovirus inhibits the inflammatory cascade by blocking Toll-like receptor (TLR) and IL-1 receptor-dependent NF-κB activation. Inhibition occurs through an interaction of the viral M45 protein with the NF-κB essential modulator (NEMO), the regulatory subunit of the IKK complex. M45 induces proteasome-independent degradation of NEMO by targeting NEMO to autophagosomes for subsequent degradation in lysosomes. We propose that the selective and irreversible degradation of a central regulatory protein by autophagy represents a new viral strategy to dampen the inflammatory response.
Author Summary
Upon viral infection cells immediately induce an innate immune response which involves the production of inflammatory cytokines. These cytokines activate specific receptors on infected and surrounding cells leading to local signal amplification as well as signal broadcasting beyond the original site of infection. Inflammatory cytokine production depends on transcription factor NF-κB, whose activity is controlled by a kinase complex that includes the NF-κB essential modulator (NEMO). In order to replicate and spread in their hosts, viruses have evolved numerous strategies to counteract innate immune defenses. In this study we identify a highly effective viral strategy to blunt the host inflammatory response: The murine cytomegalovirus M45 protein binds to NEMO and redirects it to autophagosomes, vesicular structures that deliver cytoplasmic constituents to lysosomes for degradation and recycling. By this means, the virus installs a sustained block to all classical NF-κB activation pathways, which include signaling cascades originating from pattern recognition receptors and inflammatory cytokine receptors. Redirection of an essential component of the host cell defense machinery to the autophagic degradation pathway is a previously unrecognized viral immune evasion strategy whose principle is likely shared by other pathogens.
PMCID: PMC3271075  PMID: 22319449
15.  The in silico macrophage: toward a better understanding of inflammatory disease 
Genome Medicine  2011;3(1):4.
Macrophages function as sentinel, cell-regulatory 'hubs' capable of initiating, perpetuating and contributing to the resolution of an inflammatory response, following their activation from a resting state. Highly complex and varied gene expression programs within the macrophage enable such functional diversity. To investigate how programs of gene expression relate to the phenotypic attributes of the macrophage, the development of in silico modeling methods is needed. Such models need to cover multiple scales, from molecular pathways in cell-autonomous immunity and intercellular communication pathways in tissue inflammation to whole organism response pathways in systemic disease. Here, we highlight the potential of in silico macrophage modeling as an amenable and important yet under-exploited tool in aiding in our understanding of the immune inflammatory response. We also discuss how in silico macrophage modeling can help in future therapeutic strategies for modulating both the acute protective effects of inflammation (such as host defense and tissue repair) and the harmful chronic effects (such as autoimmune diseases).
PMCID: PMC3092089  PMID: 21349141
16.  Temporal Profiling of the Coding and Noncoding Murine Cytomegalovirus Transcriptomes▿† 
Journal of Virology  2011;85(12):6065-6076.
The global transcriptional program of murine cytomegalovirus (MCMV), involving coding, noncoding, and antisense transcription, remains unknown. Here we report an oligonucleotide custom microarray platform capable of measuring both coding and noncoding transcription on a genome-wide scale. By profiling MCMV wild-type and immediate-early mutant strains in fibroblasts, we found rapid activation of the transcriptome by 6.5 h postinfection, with absolute dependency on ie3, but not ie1 or ie2, for genomic programming of viral gene expression. Evidence is also presented to show, for the first time, genome-wide noncoding and bidirectional transcription at late stages of MCMV infection.
PMCID: PMC3126304  PMID: 21471238
17.  The Activator Protein 1 Binding Motifs within the Human Cytomegalovirus Major Immediate-Early Enhancer Are Functionally Redundant and Act in a Cooperative Manner with the NF-κB Sites during Acute Infection ▿ §  
Journal of Virology  2010;85(4):1732-1746.
Human cytomegalovirus (HCMV) infection causes a rapid induction of c-Fos and c-Jun, the major subunits of activator protein 1 (AP-1), which in turn have been postulated to activate the viral immediate-early (IE) genes. Accordingly, the major IE promoter (MIEP) enhancer, a critical control region for initiating lytic HCMV infection and reactivation from the latent state, contains one well-characterized AP-1 site and a second candidate interaction site. In this study we explored the role of these AP-1 elements in the context of the infection. We first show that the distal candidate AP-1 motif binds c-Fos/c-Jun heterodimers (AP-1 complex) and confers c-Fos/c-Jun-mediated activity to a core promoter. Site-directed mutagenesis studies indicate that both AP-1 response elements are critical for 12-O-tetradecanoylphorbol-13-acetate (TPA)-enhanced MIEP activity in transient-transfection assays. In marked contrast to the results obtained with the isolated promoter, disruption of the AP-1 recognition sites of the MIEP in the context of the infectious HCMV genome has no significant influence on the expression of the MIE protein IE1 or viral replication in different cell types. Moreover, a chimeric murine CMV driven by the HCMV MIEP (hMCMV-ES) with the two AP-1 binding sites mutated is not compromised in virulence, is able to grow and disseminate to different organs of the newborn mice as efficiently as the parental virus, and is competent in reactivation. We show, however, that combined inactivation of the enhancer AP-1 and NF-κB recognition sites leads to an attenuation of the hMCMV-ES in the neonatal murine infection model, not observed when each single element is abolished. Altogether, these results underline the functional redundancy of the MIEP elements, highlighting the plasticity of this region, which probably evolved to ensure maximal transcriptional performance across many diverse environments.
PMCID: PMC3028895  PMID: 21106746
18.  Host Defense against Viral Infection Involves Interferon Mediated Down-Regulation of Sterol Biosynthesis 
PLoS Biology  2011;9(3):e1000598.
Upon infection, our immune cells produce a small protein called interferon, which in turn signals a protective response through a series of biochemical reactions that involves lowering the cells' ability to make cholesterol by targeting a gene essential for controlling the pathway for cholesterol metabolism.
Little is known about the protective role of inflammatory processes in modulating lipid metabolism in infection. Here we report an intimate link between the innate immune response to infection and regulation of the sterol metabolic network characterized by down-regulation of sterol biosynthesis by an interferon regulatory loop mechanism. In time-series experiments profiling genome-wide lipid-associated gene expression of macrophages, we show a selective and coordinated negative regulation of the complete sterol pathway upon viral infection or cytokine treatment with IFNγ or β but not TNF, IL1β, or IL6. Quantitative analysis at the protein level of selected sterol metabolic enzymes upon infection shows a similar level of suppression. Experimental testing of sterol metabolite levels using lipidomic-based measurements shows a reduction in metabolic output. On the basis of pharmacologic and RNAi inhibition of the sterol pathway we show augmented protection against viral infection, and in combination with metabolite rescue experiments, we identify the requirement of the mevalonate-isoprenoid branch of the sterol metabolic network in the protective response upon statin or IFNβ treatment. Conditioned media experiments from infected cells support an involvement of secreted type 1 interferon(s) to be sufficient for reducing the sterol pathway upon infection. Moreover, we show that infection of primary macrophages containing a genetic knockout of the major type I interferon, IFNβ, leads to only a partial suppression of the sterol pathway, while genetic knockout of the receptor for all type I interferon family members, ifnar1, or associated signaling component, tyk2, completely abolishes the reduction of the sterol biosynthetic activity upon infection. Levels of the proteolytically cleaved nuclear forms of SREBP2, a key transcriptional regulator of sterol biosynthesis, are reduced upon infection and IFNβ treatment at both the protein and de novo transcription level. The reduction in srebf2 gene transcription upon infection and IFN treatment is also found to be strictly dependent on ifnar1. Altogether these results show that type 1 IFN signaling is both necessary and sufficient for reducing the sterol metabolic network activity upon infection, thereby linking the regulation of the sterol pathway with interferon anti-viral defense responses. These findings bring a new link between sterol metabolism and interferon antiviral response and support the idea of using host metabolic modifiers of innate immunity as a potential antiviral strategy.
Author Summary
Currently, little is known about the crosstalk between the body's immune and metabolic systems that occurs after viral infection. This work uncovers a previously unappreciated physiological role for the cholesterol-metabolic pathway in protecting against infection that involves a molecular link with the protein interferon, which is made by immune cells and known to “interfere” with viral replication. We used a clinically relevant model based on mouse cytomegalovirus (CMV) infection of bone-marrow-derived cells. Upon infection these cells produce high levels of interferon as part of the innate-immune response, which we show in turn signals through the interferon receptor resulting in lowering enzyme levels on the cholesterol pathway. We observed this effect with a range of other viruses, and in each case it leads to a notable drop in the metabolites involved in the cholesterol pathway. We found that the control mechanism involves regulation by interferon of an essential transcription factor, named SREBP-2, which coordinates the gene activity of the cholesterol pathway. This mechanism may explain clinical observations of reduced cholesterol levels in patients receiving interferon treatment. Our initial investigation into how lowered cholesterol might protect against viral infection reveals that the protection is not due to a requirement of the virus for cholesterol itself but instead involves a particular side-branch of the pathway that chemically links lipids to proteins. Drugs such as statins and small interfering RNAs that block this part of the pathway are also shown to protect against CMV infection of cells in culture and in mice. This provides the first example of targeting a host metabolic pathway in order to protect against an acute infection.
PMCID: PMC3050939  PMID: 21408089
19.  Enhancerless Cytomegalovirus Is Capable of Establishing a Low-Level Maintenance Infection in Severely Immunodeficient Host Tissues but Fails in Exponential Growth▿  
Journal of Virology  2010;84(12):6254-6261.
Major immediate-early transcriptional enhancers are genetic control elements that act, through docking with host transcription factors, as a decisive regulatory unit for efficient initiation of the productive virus cycle. Animal models are required for studying the function of enhancers paradigmatically in host organs. Here, we have sought to quantitatively assess the establishment, maintenance, and level of in vivo growth of enhancerless mutants of murine cytomegalovirus in comparison with those of an enhancer-bearing counterpart in models of the immunocompromised or immunologically immature host. Evidence is presented showing that enhancerless viruses are capable of forming restricted foci of infection but fail to grow exponentially.
PMCID: PMC2876661  PMID: 20375164
20.  The mEPN scheme: an intuitive and flexible graphical system for rendering biological pathways 
BMC Systems Biology  2010;4:65.
There is general agreement amongst biologists about the need for good pathway diagrams and a need to formalize the way biological pathways are depicted. However, implementing and agreeing how best to do this is currently the subject of some debate.
The modified Edinburgh Pathway Notation (mEPN) scheme is founded on a notation system originally devised a number of years ago and through use has now been refined extensively. This process has been primarily driven by the author's attempts to produce process diagrams for a diverse range of biological pathways, particularly with respect to immune signaling in mammals. Here we provide a specification of the mEPN notation, its symbols, rules for its use and a comparison to the proposed Systems Biology Graphical Notation (SBGN) scheme.
We hope this work will contribute to the on-going community effort to develop a standard for depicting pathways and will provide a coherent guide to those planning to construct pathway diagrams of their biological systems of interest.
PMCID: PMC2878301  PMID: 20478018
21.  Construction of a large scale integrated map of macrophage pathogen recognition and effector systems 
BMC Systems Biology  2010;4:63.
In an effort to better understand the molecular networks that underpin macrophage activation we have been assembling a map of relevant pathways. Manual curation of the published literature was carried out in order to define the components of these pathways and the interactions between them. This information has been assembled into a large integrated directional network and represented graphically using the modified Edinburgh Pathway Notation (mEPN) scheme.
The diagram includes detailed views of the toll-like receptor (TLR) pathways, other pathogen recognition systems, NF-kappa-B, apoptosis, interferon signalling, MAP-kinase cascades, MHC antigen presentation and proteasome assembly, as well as selected views of the transcriptional networks they regulate. The integrated pathway includes a total of 496 unique proteins, the complexes formed between them and the processes in which they are involved. This produces a network of 2,170 nodes connected by 2,553 edges.
The pathway diagram is a navigable visual aid for displaying a consensus view of the pathway information available for these systems. It is also a valuable resource for computational modelling and aid in the interpretation of functional genomics data. We envisage that this work will be of value to those interested in macrophage biology and also contribute to the ongoing Systems Biology community effort to develop a standard notation scheme for the graphical representation of biological pathways.
PMCID: PMC2892459  PMID: 20470404
22.  Statistical Methods for Analysis of High-Throughput RNA Interference Screens 
Nature methods  2009;6(8):569-575.
RNA interference (RNAi) has become a powerful technique for reverse genetics and drug discovery and, in both of these areas, large-scale high-throughput RNAi screens are commonly performed. The statistical techniques used to analyze these screens are frequently borrowed directly from small-molecule screening; however small-molecule and RNAi data characteristics differ in meaningful ways. We examine the similarities and differences between RNAi and small-molecule screens, highlighting particular characteristics of RNAi screen data that must be addressed during analysis. Additionally, we provide guidance on selection of analysis techniques in the context of a sample workflow.
PMCID: PMC2789971  PMID: 19644458
23.  Promoting coherent minimum reporting guidelines for biological and biomedical investigations: the MIBBI project 
Nature biotechnology  2008;26(8):889-896.
The Minimum Information for Biological and Biomedical Investigations (MIBBI) project provides a resource for those exploring the range of extant minimum information checklists and fosters coordinated development of such checklists.
PMCID: PMC2771753  PMID: 18688244
24.  The Mouse Cytomegalovirus Immediate-Early 1 Gene Is Not Required for Establishment of Latency or for Reactivation in the Lungs▿  
Journal of Virology  2009;83(9):4030-4038.
The immediate-early protein IE1 of human and mouse cytomegalovirus (MCMV) is one of the first proteins expressed during the productive infection cycle and upon reactivation from latency. The CMV IE1 proteins have been found to inhibit histone deacetylases, suggesting a role in the epigenetic regulation of viral gene expression. Consequently, the IE1 protein is considered to have a profound effect on reactivation, because small amounts of IE1 may be decisive for the switch to lytic replication. Here we asked if an MCMV Δie1 mutant is able both to establish latency and to reactivate from the lungs of latently infected mice. Since the Δie1 mutant was known to be attenuated during acute infection, we first defined conditions that led to comparable levels of viral genomes during latent infection with mutant and wild-type (wt) MCMV. Viral genome copy numbers dropped considerably at the onset of the latent infection but then remained steady for both viruses even after several months. Reactivation of the Δie1 mutant and of wt MCMV from latency occurred with similar incidences in lung explant cultures at 4, 7, and 12 months postinfection. The increase in the frequency of a subset of MCMV-specific memory T cells, a possible indicator of frequent transcriptional reactivation events during latency, was in a comparable range for both viruses. Recurrence of the Δie1 virus infection in vivo could also be induced by hematoablative treatment of latently infected mice. We conclude that the ie1 gene is not essential for the establishment of latency or for the reactivation of MCMV.
PMCID: PMC2668463  PMID: 19211741
25.  High-throughput sequence analysis of variants of human cytomegalovirus strains Towne and AD169 
The Journal of General Virology  2009;90(Pt 10):2375-2380.
The genomes of commonly used variants of human cytomegalovirus (HCMV) strains Towne and AD169 each contain a substantial mutation in which a region (UL/b′) at the right end of the long unique region has been replaced by an inverted duplication of a region from the left end of the genome. Using high-throughput technology, we have sequenced HCMV strain Towne (ATCC VR-977) and confirmed the presence of two variants, one exhibiting the replacement in UL/b′ and the other intact in this region. Both variants are mutated in genes RL13, UL1, UL40, UL130, US1 and US9. We have also sequenced a novel AD169 variant (varUC) that is intact in UL/b′ except for a small deletion that affects genes UL144, UL142, UL141 and UL140. Like other AD169 variants, varUC is mutated in genes RL5A, RL13, UL36 and UL131A. A subpopulation of varUC contains an additional deletion affecting genes IRS1, US1 and US2.
PMCID: PMC2885757  PMID: 19553388

Results 1-25 (52)