RNA polymerase II stalling promotes nucleosome occlusion and pTEFb recruitment to drive immortalization by Epstein-Barr virus

Epstein-Barr virus (EBV) immortalizes resting B-cells and is a key etiologic agent in the development of numerous cancers. The essential EBV-encoded protein EBNA 2 activates the viral C promoter (Cp) producing a message of ~120 kb that is differentially spliced to encode all EBNAs required for immortalization. We have previously shown that EBNA 2-activated transcription is dependent on the activity of the RNA polymerase II (pol II) C-terminal domain (CTD) kinase pTEFb (CDK9/cyclin T1). We now demonstrate that Cp, in contrast to two shorter EBNA 2-activated viral genes (LMP 1 and 2A), displays high levels of promoter-proximally stalled pol II despite being constitutively active. Consistent with pol II stalling, we detect considerable pausing complex (NELF/DSIF) association with Cp. Significantly, we observe substantial Cp-specific pTEFb recruitment that stimulates high-level pol II CTD serine 2 phosphorylation at distal regions (up to +75 kb), promoting elongation. We reveal that Cp-specific pol II accumulation is directed by DNA sequences unfavourable for nucleosome assembly that increase TBP access and pol II recruitment. Stalled pol II then maintains Cp nucleosome depletion. Our data indicate that pTEFb is recruited to Cp by the bromodomain protein Brd4, with polymerase stalling facilitating stable association of pTEFb. The Brd4 inhibitor JQ1 and the pTEFb inhibitors DRB and Flavopiridol significantly reduce Cp, but not LMP1 transcript production indicating that Brd4 and pTEFb are required for Cp transcription. Taken together our data indicate that pol II stalling at Cp promotes transcription of essential immortalizing genes during EBV infection by (i) preventing promoter-proximal nucleosome assembly and ii) necessitating the recruitment of pTEFb thereby maintaining serine 2 CTD phosphorylation at distal regions

Firm technological responses to regulatory changes: A longitudinal study in the Le Mans Prototype racing

Despite the critical role of regulations on competition and innovation, little is known about firm responses and related effects on performance under regulatory contingencies that are permissive or restrictive. By longitudinally investigating hybrid cars competing in the Le Mans Prototype racing (LMP1), we counter-intuitively suggest that permissive regulations increase technological uncertainty and thus decrease the firms’ likelihood of shifting their technological trajectory, while restrictive regulations lead to the opposite outcome. Further, we suggest that permissive regulations favour firms that innovate their products by sequentially upgrading core and peripheral subsystems, while restrictive regulations—in the long term— favour firms upgrading them simultaneously. Implications for theory and practice are discussed

Small-Sample Behavior of Novel Phase I Cancer Trial Designs

Novel dose-finding designs, using estimation to assign the best estimated maximum- tolerated-dose (MTD) at each point in the experiment, most commonly via Bayesian techniques, have recently entered large-scale implementation in Phase I cancer clinical trials. We examine the small-sample behavior of these "Bayesian Phase I" (BP1) designs, and also of non-Bayesian designs sharing the same main "long-memory" traits (hereafter: LMP1s). For all LMP1s examined, the number of cohorts treated at the true MTD (denoted here as n*) was highly variable between numerical runs drawn from the same toxicity-threshold distribution, especially when compared with "up-and-down" (U&D) short-memory designs. Further investigation using the same set of thresholds in permuted order, produced a nearly-identical magnitude of variability in n*. Therefore, this LMP1 behavior is driven by a strong sensitivity to the order in which toxicity thresholds appear in the experiment. We suggest that the sensitivity is related to LMP1's tendency to "settle" early on a specific dose level - a tendency caused by the repeated likelihood-based "winner-takes-all" dose assignment rule, which grants the early cohorts a disproportionately large influence upon experimental trajectories. Presently, U&D designs offer a simpler and more stable alternative, with roughly equivalent MTD estimation performance. A promising direction for combining the two approaches is briefly discussed (note: the '3+3' protocol is not a U&D design).Comment: Somewhat modified version of the version accepted pending final modifications at Clinical Trials. The supplement is in the bac

N-acetylcysteine (NAC) ameliorates Epstein-Barr virus latent membrane protein 1 induced chronic inflammation

Chronic inflammation results when the immune system responds to trauma, injury or infection and the response is not resolved. It can lead to tissue damage and dysfunction and in some cases predispose to cancer. Some viruses (including Epstein-Barr virus (EBV)) can induce inflammation, which may persist even after the infection has been controlled or cleared. The damage caused by inflammation, can itself act to perpetuate the inflammatory response. The latent membrane protein 1 (LMP1) of EBV is a pro-inflammatory factor and in the skin of transgenic mice causes a phenotype of hyperplasia with chronic inflammation of increasing severity, which can progress to pre-malignant and malignant lesions. LMP1 signalling leads to persistent deregulated expression of multiple proteins throughout the mouse life span, including TGFα S100A9 and chitinase-like proteins. Additionally, as the inflammation increases, numerous chemokines and cytokines are produced which promulgate the inflammation. Deposition of IgM, IgG, IgA and IgE and complement activation form part of this process and through genetic deletion of CD40, we show that this contributes to the more tissue-destructive aspects of the phenotype. Treatment of the mice with N-acetylcysteine (NAC), an antioxidant which feeds into the body’s natural redox regulatory system through glutathione synthesis, resulted in a significantly reduced leukocyte infiltrate in the inflamed tissue, amelioration of the pathological features and delay in the inflammatory signature measured by in vivo imaging. Reducing the degree of inflammation achieved through NAC treatment, had the knock on effect of reducing leukocyte recruitment to the inflamed site, thereby slowing the progression of the pathology. These data support the idea that NAC could be considered as a treatment to alleviate chronic inflammatory pathologies, including post-viral disease. Additionally, the model described can be used to effectively monitor and accurately measure therapies for chronic inflammation

Upregulation of Id1 by Epstein-Barr Virus-encoded LMP1 confers resistance to TGFβ-mediated growth inhibition

BACKGROUND: Epstein-Barr virus (EBV)-encoded LMP1 protein is commonly expressed in nasopharyngeal carcinoma (NPC). LMP1 is a prime candidate for driving tumourigenesis given its ability to activate multiple signalling pathways and to alter the expression and activity of variety of downstream targets. Resistance to TGFβ-mediated cytostasis is one of the growth transforming effects of LMP1. Of the downstream targets manipulated by LMP1, the induction of Id1 and inactivation of Foxo3a appear particularly relevant to LMP1-mediated effects. Id1, a HLH protein is implicated in cell transformation and plays a role in cell proliferation, whilst Foxo3a, a transcription factor controls cell integrity and homeostasis by regulating apoptosis. The mechanism(s) by which LMP1 induces these effects have not been fully characterised. RESULTS: In this study, we demonstrate that the ability of LMP1 to induce the phosphorylation and inactivation of Foxo3a is linked to the upregulation of Id1. Furthermore, we show that the induction of Id1 is essential for the transforming function of LMP1 as over-expression of Id1 increases cell proliferation, attenuates TGFβ-SMAD-mediated transcription and renders cells refractory to TGFβ-mediated cytostasis. Id1 silencing in LMP1-expressing epithelial cells abolishes the inhibitory effect of LMP1 on TGFβ-mediated cell growth arrest and reduces the ability of LMP1 to attenuate SMAD transcriptional activity. In response to TGFβ stimulation, LMP1 does not abolish SMAD phosphorylation but inhibits p21 protein expression. In addition, we found the induction of Id1 in LMP1-expressing cells upon stimulation by TGFβ. We provide evidence that LMP1 suppresses the transcriptional repressor ATF3, possibly leading to the TGFβ-induced Id1 upregulation. CONCLUSION: The current data provide novel information regarding the mechanisms by which LMP1 suppresses TGFβ-induced cytostasis, highlighting the importance of Id1 in LMP1 mediated cell transformatio

Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors

Viral hijacking of cellular metabolism.

This review discusses the current state of the viral metabolism field and gaps in knowledge that will be important for future studies to investigate. We discuss metabolic rewiring caused by viruses, the influence of oncogenic viruses on host cell metabolism, and the use of viruses as guides to identify critical metabolic nodes for cancer anabolism. We also discuss the need for more mechanistic studies identifying viral proteins responsible for metabolic hijacking and for in vivo studies of viral-induced metabolic rewiring. Improved technologies for detailed metabolic measurements and genetic manipulation will lead to important discoveries over the next decade

NF-κB and IRF7 Pathway Activation by Epstein-Barr Virus Latent Membrane Protein 1

The principal Epstein-Barr virus (EBV) oncoprotein, Latent Membrane Protein 1 (LMP1), is expressed in most EBV-associated human malignancies. LMP1 mimics CD40 receptor signaling to provide infected cells with constitutive NF-κB, MAP kinase, IRF7, and PI3 kinase pathway stimulation. EBV-transformed B-cells are particularly dependent on constitutive NF-κB activity, and rapidly undergo apoptosis upon NF-κB blockade. Here, we review LMP1 function, with special attention to current understanding of the molecular mechanisms of LMP1-mediated NF-κB and IRF7 pathway activation. Recent advances include the elucidation of transmembrane motifs important for LMP1 trafficking and ligand-independent signaling, analysis of genome-wide LMP1 gene targets, and the identification of novel cell proteins that mediate LMP1 NF-κB and IRF7 pathway activation

Deciphering the role of Epstein-Barr virus in the pathogenesis of T and NK cell lymphoproliferations

Epstein-Barr virus (EBV) is a highly successful herpesvirus, colonizing more than 90% of the adult human population worldwide, although it is also associated with various malignant diseases. Primary infection is usually clinically silent, and subsequent establishment of latency in the memory B lymphocyte compartment allows persistence of the virus in the infected host for life. EBV is so markedly B-lymphotropic when exposed to human lymphocytes in vitro that the association of EBV with rare but distinct types of T and NK cell lymphoproliferations was quite unexpected. Whilst relatively rare, these EBV-associated T and NK lymphoproliferations can be therapeutically challenging and prognosis for the majority of patients is dismal. In this review, we summarize the current knowledge on the role of EBV in the pathogenesis of these tumours, and the implications for treatment. \ud \u