Mechanism of activation of the BNLF2a immune evasion gene of Epstein-Barr virus by Zta

The human gamma herpes virus Epstein–Barr virus (EBV) exploits multiple routes to evade the cellular immune response. During the EBV lytic replication cycle, viral proteins are expressed that provide excellent targets for recognition by cytotoxic T cells. This is countered by the viral BNLF2a gene. In B cells during latency, where BNLF2a is not expressed, we show that its regulatory region is embedded in repressive chromatin. The expression of BNLF2a mirrors the expression of a viral lytic cycle transcriptional regulator, Zta (BZLF1, EB1, ZEBRA), in B cells and we propose that Zta plays a role in up-regulating BNLF2a. In cells undergoing EBV lytic replication, we identified two distinct regions of interaction of Zta with the chromatin-associated BNLF2a promoter. We identify five potential Zta-response elements (ZREs) in the promoter that are highly conserved between virus isolates. Zta binds to these elements in vitro and activates the expression of the BNLF2a promoter in both epithelial and B cells. We also found redundancy amongst the ZREs. The EBV genome undergoes a biphasic DNA methylation cycle during its infection cycle. One of the ZREs contains an integral CpG motif. We show that this can be DNA methylated during EBV latency and that both Zta binding and promoter activation are enhanced by its methylation. In summary, we find that the BNLF2a promoter is directly targeted by Zta and that DNA methylation within the proximal ZRE aids activation. The implications for regulation of this key viral gene during the reactivation of EBV from latency are discussed

Epstein-Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression

Lytic replication of the human gamma herpes virus Epstein-Barr virus (EBV) is an essential prerequisite for the spread of the virus. Differential regulation of a limited number of cellular genes has been reported in B-cells during the viral lytic replication cycle. We asked whether a viral bZIP transcription factor, Zta (BZLF1, ZEBRA, EB1), drives some of these changes. Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome. Using sensitive transcriptome analyses we identified 2263 cellular genes whose expression is significantly changed during the EBV lytic replication cycle. Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites. This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements. We show that a synthetic Zta binding element confers Zta regulation at a distance and that distal Zta binding sites from cellular genes can confer Zta-mediated regulation on a heterologous promoter. This leads us to propose that Zta directly reprograms the expression of cellular genes through distal elements

Identification, characterisation and expression analysis of natural killer receptor genes in Chlamydia pecorum infected koalas (Phascolarctos cinereus)

BACKGROUND: Koalas (Phascolarctos cinereus), an iconic Australian marsupial, are being heavily impacted by the spread of Chlamydia pecorum, an obligate intracellular bacterial pathogen. Koalas vary in their response to this pathogen, with some showing no symptoms, while others suffer severe symptoms leading to infertility, blindness or death. Little is known about the pathology of this disease and the immune response against it in this host. Studies have demonstrated that natural killer (NK) cells, key components of the innate immune system, are involved in the immune response to chlamydial infections in humans. These cells can directly lyse cells infected by intracellular pathogens and their ability to recognise these infected cells is mediated through NK receptors on their surface. These are encoded in two regions of the genome, the leukocyte receptor complex (LRC) and the natural killer complex (NKC). These two families evolve rapidly and different repertoires of genes, which have evolved by gene duplication, are seen in different species. METHODS: In this study we aimed to characterise genes belonging to the NK receptor clusters in the koala by searching available koala transcriptomes using a combination of search methods. We developed a qPCR assay to quantify relative expression of four genes, two encoded within the NK receptor cluster (CLEC1B, CLEC4E) and two known to play a role in NK response to Chalmydia in humans (NCR3, PRF1). RESULTS: We found that the NK receptor repertoire of the koala closely resembles that of the Tasmanian devil, with minimal genes in the NKC, but with lineage specific expansions in the LRC. Additional genes important for NK cell activity, NCR3 and PRF1, were also identified and characterised. In a preliminary study to investigate whether these genes are involved in the koala immune response to infection by its chlamydial pathogen, C. pecorum, we investigated the expression of four genes in koalas with active chlamydia infection, those with past infection and those without infection using qPCR. This analysis revealed that one of these four, CLEC4E, may be upregulated in response to chlamydia infection. CONCLUSION: We have characterised genes of the NKC and LRC in koalas and have discovered evidence that one of these genes may be upregulated in koalas with chlamydia, suggesting that these receptors may play a role in the immune response of koalas to chlamydia infection

Induction of interferon-stimulated genes on the IL-4 response axis by Epstein-Barr virus infected human b cells; relevance to cellular transformation.

Epstein-Barr virus (EBV) is an oncogenic virus that is associated with the pathogenesis of several human lymphoid malignancies, including Hodgkin's lymphoma. Infection of normal resting B cells with EBV results in activation to lymphoblasts that are phenotypically similar to those generated by physiological stimulation with CD40L plus IL-4. One important difference is that infection leads to the establishment of permanently growing lymphoblastoid cell lines, whereas CD40L/IL-4 blasts have finite proliferation lifespans. To identify early events which might later determine why EBV infected blasts go on to establish transformed cell lines, we performed global transcriptome analyses on resting B cells and on EBV and CD40L/IL-4 blasts after 7 days culture. As anticipated there was considerable overlap in the transcriptomes of the two types of lymphoblasts when compared to the original resting B cells, reflecting common changes associated with lymphocyte activation and proliferation. Of interest to us was a subset of 255 genes that were differentially expressed between EBV and CD40L/IL-4 blasts. Genes which were more highly expressed in EBV blasts were substantially and significantly enriched for a set of interferon-stimulated genes which on further in silico analyses were found to be repressed by IL-4 in other cell contexts and to be up-regulated in micro-dissected malignant cells from Hodgkin's lymphoma biopsies when compared to their normal germinal center cell counterparts. We hypothesized that EBV and IL-4 were targeting and discordantly regulating a common set of genes. This was supported experimentally in our B cell model where IL-4 stimulation partially reversed transcriptional changes which follow EBV infection and it impaired the efficiency of EBV-induced B cell transformation. Taken together, these data suggest that the discordant regulation of interferon and IL-4 pathway genes by EBV that occurs early following infection of B cells has relevance to the development or maintenance of an EBV-associated malignancy

Loss of functional pRB is not a ubiquitous feature of B-cell malignancies

Human cancers frequently sustain genetic mutations that alter the function of their G1 cell cycle control check point. These include changes to the retinoblastoma gene and to the genes that regulate its phosphorylation, such as the cyclin-dependent kinase inhibitor p16(INK4a). Altered expression of retinoblastoma protein (pRb) is associated with non-Hodgkin's lymphoma, particularly centroblastic and Burkitt's lymphomas. pRb is expressed in normal B-cells and its regulatory phosphorylation pathway is activated in response to a variety of stimuli. Since human B-lymphoma-derived cell lines are often used as in vitro model systems to analyse the downstream effects of signal transduction, we examined the functional status of pRb in a panel of human B-cell lines. We identified eleven cell lines which express the hyperphosphorylated forms of pRb. Furthermore, we suggest that the pRb protein appears to be functional in these cell lines

Modulation of NKp30- and NKp46-Mediated Natural Killer Cell Responses by Poxviral Hemagglutinin

Natural killer (NK) cells are an important element in the immune defense against the orthopox family members vaccinia virus (VV) and ectromelia virus (ECTV). NK cells are regulated through inhibitory and activating signaling receptors, the latter involving NKG2D and the natural cytotoxicity receptors (NCR), NKp46, NKp44 and NKp30. Here we report that VV infection results in an upregulation of ligand structures for NKp30 and NKp46 on infected cells, whereas the binding of NKp44 and NKG2D was not significantly affected. Likewise, infection with ectromelia virus (ECTV), the mousepox agent, enhanced binding of NKp30 and, to a lesser extent, NKp46. The hemagglutinin (HA) molecules from VV and ECTV, which are known virulence factors, were identified as novel ligands for NKp30 and NKp46. Using NK cells with selectively silenced NCR expression and NCR-CD3ζ reporter cells, we observed that HA present on the surface of VV-infected cells, or in the form of recombinant soluble protein, was able to block NKp30-triggered activation, whereas it stimulated the activation through NKp46. The net effect of this complex influence on NK cell activity resulted in a decreased NK lysis susceptibility of infected cells at late time points of VV infection when HA was expression was pronounced. We conclude that poxviral HA represents a conserved ligand of NCR, exerting a novel immune escape mechanism through its blocking effect on NKp30-mediated activation at a late stage of infection

The Natural Cytotoxicity Receptors in Health and Disease

The Natural Cytotoxicity Receptors (NCRs), NKp46, NKp44, and NKp30, were some of the first human activating Natural Killer (NK) cell receptors involved in the non-MHC-restricted recognition of tumor cells to be cloned over 20 years ago. Since this time many host- and pathogen-encoded ligands have been proposed to bind the NCRs and regulate the cytotoxic and cytokine-secreting functions of tissue NK cells. This diverse set of NCR ligands can manifest on the surface of tumor or virus-infected cells or can be secreted extracellularly, suggesting a remarkable NCR polyfunctionality that regulates the activity of NK cells in different tissue compartments during steady state or inflammation. Moreover, the NCRs can also be expressed by other innate and adaptive immune cell subsets under certain tissue conditions potentially conferring NK recognition programs to these cells. Here we review NCR biology in health and disease with particular reference to how this important class of receptors regulates the functions of tissue NK cells as well as confer NK cell recognition patterns to other innate and adaptive lymphocyte subsets. Finally, we highlight how NCR biology is being harnessed for novel therapeutic interventions particularly for enhanced tumor surveillance

NK cells and cancer: you can teach innate cells new tricks

Natural killer (NK) cells are the prototype innate lymphoid cells endowed with potent cytolytic function that provide host defence against microbial infection and tumours. Here, we review evidence for the role of NK cells in immune surveillance against cancer and highlight new therapeutic approaches for targeting NK cells in the treatment of cancer