European EBV meeting 2015

Karolinska Institutet

Department of Microbiology, Tumor and Cell Biology (MTC)

June 15-16, 2015

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Program

Monday, June 15th 2015

8.00 – 9.00 Registration, coffee, tea

9.00 - 9.10 Welcome and opening announcements

9.10 – 10.45 Session 1: Gene expression and regulation

The session chairs: Elena Kashuba & Ingemar Ernberg 9.10 – 9.55 Key-note lecture

EBV infection, NF-kB activation, and lymphomas Elliott Kieff

9.55 Superior B cell transformation ability of type 1 EBV Paul Farrell

10.10 Cellular reprogramming by EBV transcription factors is mediated by large-scale reorganisation of active chromatin hubs David Wood

10.25 Identification of a viral pre-initiation complex (vPIC) involved in EBV late gene expression Henri Gruffat

10.40 – 11.10 Refreshments

11.10 – 13.00 Session 2: Protein functions

The session chairs: Alison Sinclair & Michiel Pegtel 11.10 The EBV nuclear antigen leader protein is essential for B cell transformation

but does not displace SP100 from PML during infection Agnieszka Szymula

11.25 BHRF1 inhibits apoptosis via binding to multiple pro-death BH3-only proteins Leah Fitzsimmons

11.40 LMP2A of EBV perturbs the expression of cellular receptors at the cell surface in tumor derived epithelial cells Liudmila Matskova

11.55 Epstein Barr virus transcription factor ZTA acts through distal regulatory elements to directly control cellular gene expression Kay Osborn

12.10 - 12.55 Key-note lecture How do the EBNA3s regulate host transcription and what are the consequences Martin Allday

13.00 – 14.00 Buffet lunch

14.00 Epstein-Barr virus EBNA3A and EBNA3C together induce expression of the oncogenic MICRORNA cluster MIR-221/MIR-222 and ablate expression of its target p57KIP2 Quentin Bazot

14.15 EBNA-binding proteins and their role in cell transformation Elena Kashuba

14.30 -16.45 Session 3: Pathogenesis

The session chairs: Michelle West & Rob White 14.30 Epstein-Barr virus MIRNAS attenuate the cellular response to type I

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interferon Emmanuel Wiertz

14.45 Cell type specific modulation of BCL6 expression by interferons in cell lines derived from human hematopoietic malignancies Daniel Salamon

15.00 Exosomal-sorting of 5’PPP-EBER1 RNA evades cytosolic sensing in latent EBV-infected B cells but supports recognition by dendritic cells Niala Masoumi

15.15 Integrin Α9 gene promoter is hypermethylated and downregulated in nasopharyngeal carcinoma Imran Nawaz

15.30 Expression of chemokine receptors CCR1 and CCR2 from the chromosome instability region at human 3p21.31 is impaired in EBV-carrying Burkitt’s lymphoma cell lines Irina Kholodnyuk Holodnuka

15.45 -16.15 Refreshments

16.15 The role of oxidative stress in LMP1-induced chronic inflammation and carcinogenesis in vivo Xiao Gao

16.30 CRISPR/CAS9-mediated genome editing of herpesviruses limits productive and latent infections Robert Jan Lebbink

16.45 – 17.30 Key-note lecture Escape from latency – a proposal Wolfgang Hammerschmidt

18.00

Dinner at restaurant Königs

Tuesday, June 16th 2015

8.30 – 9.00 Coffee, tea

9.00 – 9.25 Dynamics of EBV-carrying B-cells in a modern representation of Waddingtons landscape Ingemar Ernberg

9.25 – 10.40 Session 4: Immunology

The session chairs: Teresa Frisan & Janos Minarovits 9.25 Study of surface antigenic and secretory determinants of EBV latency III B

cells that can modulate the anti-EBV T-cell response Héloïse Auclair

9.40 EBV immune evasion of NK cell killing: virus infected B cells are protected against NK cell lysis during late stage lytic cycle Luke Williams

9.55. Exosomal-sorting of 5’PPP-EBER1 RNA evades cytosolic sensing in latent EBV-infected B cells but supports recognition by dendritic cells Michiel Pegtel

10.10 The missing link in EBV immune evasion: the BDLF3 gene induces ubiquitination and degradation of MHC class I and MHC class II Laura Quinn

10.25 A novel viral immune evasion molecule interferes with antigen presentation during the late phase of Epstein-Barr virus infection

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Anna Gram

10.40 – 11.10 Refreshments

11.10 – 13.00 Session 5: Therapy and disease models

The session chairs: Evelyne Manet & Paul Farrell 11.10 Malignant lymphoma in patients before and after the introduction of

combination antiretroviral therapy (c-art) Sven Grutzmeier

11.25 EPSTEIN-BARR VIRUS REACTIVATION FOLLOWING STEM CELL transplantation is associated with the early emergence of circulating CD27+ memory B cells Andrew Bell

11.40 The effectiveness of combined antiviral therapy in chronic mononucleosis caused by Epstein-Barr virus Dmitry Maltsev

11.55 Modeling EBV latency III driven tumorigenesis in mice Thomas Sommermann

12.10 – 12.55 Key-note lecture Interactions of viruses with genetic modifications in carcinogenesis – special implications for the Epstein-Barr virus system Harald zur Hausen

13.00 – 14.00 Buffet lunch

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Session 1: Gene expression and regulation

Key-note lecture

EBV infection, NF-kB activation, and Lymphomas

Elliott Kieff

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SUPERIOR B CELL TRANSFORMATION ABILITY OF TYPE 1 EBV

Farrell PJ (1), Correia P (1), Correia S (1), Tzellos S (1), West MJ (2), Elgueta Karstegl C (1)

(1) Section of Virology, Imperial College London, UK; (2) School of Life Sciences,

University of Sussex, UK Analysis of 83 EBV genome sequences confirmed that the type 1/type 2 classification is the main form of natural variation of EBV (Palser et al 2015). It is determined almost exclusively by EBNA2 and EBNA3 genes in the regions that were sequenced. There is strong genetic linkage between the EBNA2 and EBNA3 genes; 81/83 sequences show linkage of the EBNA2 and EBNA3 types, only 2/83 have type 1 EBNA2 with type 2 EBNA3 genes. We previously showed that conversion of a single amino acid (S442D) in type 2 EBNA2 to the aspartate found in type 1 EBNA2 was sufficient to increase its function in an EREB2.5 LCL growth maintenance assay to the type 1 EBNA2 level. We have now recombined the same alleles of EBNA2 into B95-8 BAC EBV and tested transformation and growth of resulting LCLs. In this assay the S442D change gives a significant but much lower enhancement of B cell transformation efficiency and the resulting LCLs also grow more slowly. The results suggest a functional type-specific interdependence of EBNA2 and EBNA3 proteins. The cell protein BS69 (ZMYND11) can regulate transcription activation by EBNA2 (Ansieau, 2002). BS69 binds to EBNA2 adjacent to the S442D polymorphism and also has consensus binding sites in type 1 EBNA3A and EBNA3C but not in type 2 EBNA3A and 3C. The superior transforming ability of type 1 EBV is thus likely to involve BS69 and both EBNA2 and EBNA3 proteins, consistent with their genetic linkage.

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CELLULAR REPROGRAMMING BY EBV TRANSCRIPTION FACTORS IS MEDIATED BY LARGE-SCALE REORGANISATION OF ACTIVE CHROMATIN

HUBS

Wood CD (1), McClellan MJ (1), Andrews S (2), West MJ (1) (1) University of Sussex, Brighton, UK; (2) The Babrham Institute, Cambridge, UK The Epstein-Barr virus transcription factors EBNA2, 3A, 3B and 3C are the key drivers of cellular reprogramming in EBV-immortalised cells. EBNA2 upregulates numerous growth control genes including MYC, whilst EBNA3A and 3C silence key targets including the pro-apoptotic gene BCL2L11 (Bim). The mechanism of MYC activation by EBNA2 was unknown until recent ChIP-seq experiments identified EBNA2-bound MYC enhancers at -168/-186kb, -428kb, and -556kb upstream from the promoter. We have identified additional EBNA2 binding sites 450kb, 570kb, 900kb and 1.8kb downstream of MYC. Using 4C-seq we examined the effect of EBNA2 binding on enhancer-promoter interactions at MYC and found that EBNA2 induces substantial directional reorganisation of the locus over a 3Mb region, reducing downstream interactions while simultaneously increasing upstream interactions to activate MYC transcription. Key increased interactions were observed at the -168/-186kb, -428kb, and -556kb enhancers in the presence of EBNA2. Investigating how EBNA 3A and 3C silence Bim transcription, we found that these proteins bind to long-range elements in a region 400kb downstream and upstream from the promoter comprising a chromatin hub including the upstream ACOXL gene promoter. Chromatin conformation analysis revealed that EBNA3A and 3C binding to these elements disrupts this hub, inhibiting interactions between the Bim-ACOXL promoters and their enhancers to promote transcriptional repression. These data indicate that a critical component of EBV-mediated cellular reprogramming involves large-scale reorganisation of host genome chromatin architecture.

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IDENTIFICATION OF A VIRAL PRE-INITIATION COMPLEX (vPIC) INVOLVED IN EBV LATE GENE EXPRESSION

Gruffat H (1-5), Aubry V (1-5), Mure F (1-5), Mariamé B (6), Deschamps T (1-5)

and Manet E (1-5)

(1) CIRI (Centre International de Recherche en Infectiologie), Oncogenic herpesviruses Team, Université de Lyon, 69364 Lyon, France; (2) Inserm, U1111, 69364 Lyon, France; (3) Ecole Normale Supérieure de Lyon, 69364 Lyon, France; (4) Université Lyon 1, Centre International de Recherche en Infectiologie, 69364 Lyon, France; (5) CNRS, UMR5308, 69364 Lyon, France; (6) CNRS UMS3039

ITAV - Institut des Technologies Avancées en sciences du Vivant, Team ANCHOR, 1, place Pierre Potier - Centre Le Potier - Cancéropôle Entrée B - BP

50624, 31106 TOULOUSE Cedex 1 France During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that has three general stages: immediate early (IE), early (E), and late (L). Promoter complexity differs strikingly between IE/E genes and L genes. IE and E promoters contain cis-regulating sequences upstream of a TATA box whereas L promoters usually comprise a unique cis element. In the case of the gamma-herpesviruses, this element is most often a TATT motif found in the position where the consensus TATA box of eukaryotic promoters usually localizes. In eukaryotes, the first step of transcriptional activation is the formation of a permissive chromatin, which allows assembly, at the core promoter, of the pre-initiation complex (PIC) formed around the TATA-binding protein. We have previously shown that EBV encodes a protein, called BcRF1, which has structural homology with the TATA-binding protein and interacts specifically with the TATT box. However, although necessary for the expression of the L genes, BcRF1 is not sufficient, suggesting that other viral proteins are also required. We have now identified 5 viral proteins in addition to BcRF1 necessary for the expression of the late genes. When expressed together the six viral proteins form a complex that we called vPIC (viral specific Pre-Initiation Complex) able to recruit the cellular RNAP-II, and sufficient for the induction of late gene expression in a transient assay. Interestingly, the EBV vPIC has homologs in beta- and gamma-herpesviruses, but not in alpha-herpesviruses.

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Session 2: Protein functions

THE EBV NUCLEAR ANTIGEN LEADER PROTEIN IS ESSENTIAL FOR B CELL TRANSFORMATION BUT DOES NOT DISPLACE SP100 FROM PML

DURING INFECTION

Szymula A, White R

Imperial College London, London, W2 1PG, UK The EBV nuclear antigen leader protein (EBNA-LP) is expressed, along with EBNA-2 early during B cell transformation. While EBNA2 is essential, little is known about the role or mechanism of action of EBNA-LP in transformation, although EBV mutants lacking the carboxy-terminal domain of EBNA-LP (exons Y1 and Y2), are much less transforming than wild-type viruses. It has been also previously shown that EBNA-LP interacts with SP100 protein and therefore may act as a co-activator of EBNA-2. We have generated an EBNA-LP knockout EBV (LPKO) and its revertant, and used it to infect primary B cells. Our observations suggested that- in contrast to an EBNA2 knockout -LPKO initially activates CD19 positive primary B cells but fails to sustain transformation. Using flow cytometry, we found that some B cells infected with LPKO are able to proliferate in the first ten days after infection. However, the number of dividing cells is significantly lower than in wild type virus infection. Cell cycle analysis indicates that more LPKO-infected B cells remain in G0/1, and we were not able to establish LCLs after infection of EBV-negative B cells, even on feeder cells. Preliminary immunofluorescence microscopy experiments have shown that two days after infection with LPKO, SP100 is displaced from ND10 domains to the cytoplasm, just as it is by WT EBV infection. However, seven days after infection with WT virus, SP100 has largely relocalised to foci in the nucleus, whereas in LPKO-infected cells, it remains in the cytoplasm.

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BHRF1 INHIBITS APOPTOSIS VIA BINDING TO MULTIPLE PRO-DEATH BH3-ONLY PROTEINS

Fitzsimmons L (1), Cartlidge R (1), Rowe M (1) and Kelly GL (2)

(1) University of Birmingham, School of Cancer Sciences, Edgbaston, B15 2TT, UK; (2) The Walter and Eliza Hall Institute of Medical Research, Royal Parade,

Parkville Victoria 3052, Australia Following its discovery in Burkitt lymphoma cells EBV was the first virus shown to be etiologically associated with human cancer; however the precise pathogenic role of EBV in BL is poorly understood. EBV-positive BLs can be divided into two subsets, based on their viral gene expression. In most, only the genome maintenance protein, EBNA1, and a number of non-coding RNAs are present (Latency I), but in a sub-set, known as Wp-restricted tumours a broader range of viral antigens is expressed. Wp-restricted BL (Wp-BL) cell lines are highly resistant to apoptosis and we suspect that patients harbouring Wp-restricted tumours may be resistant to treatment and are therefore likely to have a very poor prognosis. EBV expresses high levels of the Bcl-2 homologue BHRF1 in Wp-BL cells which is potently anti-apoptotic and we are now investigating the molecular mechanism by which BHRF1 confers protection to Wp-BL cells. We have used a series of mutant BHRF1 proteins to profile the interactions of BHRF1 with the cellular Bcl-2 family members and confirmed that it is able to interact with and therefore inhibit several pro-apoptotic Bcl-2 family proteins in response to a range of apoptosis-inducers.

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LMP2A OF EBV PERTURBS THE EXPRESSION OF CELLULAR RECEPTORS AT THE CELL SURFACE IN TUMOR DERIVED EPITHELIAL CELLS

Matskova L (1), Xiaoying Z (1), Zhang Z (2) and Ernberg I (1)

(1) MTC KI, Stockholm, Sweden; (2) Guangxi Medical University, Ninning, China

Epstein-Barr virus (EBV) encoded Latent Membrane Protein 2A (LMP2A) mRNA is frequently detected in nasopharyngeal carcinoma (NPC) tissues. LMP2A mediates a constitutive activation of the Shb-Syk-PI3K-Akt pathway in transfected epithelial cell lines established from NPC tumors, mimicking cellular surviving and mitogenic stimuli. LMP2A also modulate migration and invasion in NPC cells. Several molecular mechanisms of LMP2A-induced migration have been demonstrated, like clustering of integrin beta 4 (ITGB4) at the cell surface. Here we report LMP2A mediated perturbation of lipid metabolism in NPC cells, which collectively affects localization of a number of cellular receptors at the cell surface, relevant for the role of LMP2A in NPC tumorigenesis.

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EPSTEIN BARR VIRUS TRANSCRIPTION FACTOR ZTA ACTS THROUGH DISTAL REGULATORY ELEMENTS TO DIRECTLY CONTROL CELLULAR

GENE EXPRESSION

Osborn K (1), Ramasubramanyan S (1), Al-Mohammad R (1), Perez-Fernandez IBN (1), Zuo J (2), Balan N (1), Godfrey A (1), Patel H (3), Peters P (3),

Rowe M (2), Jenner RG (4), Sinclair AJ (1)

(1) School of Life Sciences, University of Sussex, Brighton BN1 9QG, UK; (2) School of Cancer Sciences, The University of Birmingham, Birmingham, B15 2TT

UK; (3) Cancer Research UK London Research Institute, 44 Lincoln's Inn FieldsLondon WC2A 3LY, UK; (4) UCL Cancer Institute and MRC Centre for

Medical Molecular Virology, Paul O'Gorman Building, University College London, London W1CE 6BT, UK

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.

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Key-note lecture

How do the EBNA3s regulate host transcription and what are the consequences

Martin Allday

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EPSTEIN-BARR VIRUS EBNA3A AND EBNA3C TOGETHER INDUCE EXPRESSION OF THE ONCOGENIC MICRORNA CLUSTER MIR-221/MIR-222

AND ABLATE EXPRESSION OF ITS TARGET p57KIP2

Bazot Q, Paschos K, Skalska L, Parker G and Allday MJ

Section of Virology, Department of Medicine, Imperial College London, London, W2 1PG, UK

We show that a host-encoded primary non-coding RNA (pri-miR) and the corresponding microRNA (miR) cluster – widely reported to have cell transformation-associated activity – are regulated by EBNA3A and EBNA3C. Utilizing a variety of EBV-transformed lymphoblastoid cell lines (LCLs) carrying either knockout-, revertant- or conditional-EBV recombinants, it was possible to demonstrate unambiguously that EBNA3A and EBNA3C are both required for transactivation of the oncogenic miR-221/miR-222 cluster that is expressed at high levels in multiple human tumours – including lymphoma/leukemia. ChIP, ChIP-seq and chromatin conformation capture (CCC) analyses indicate that this activation results from direct targeting of both EBV proteins to an enhancer located within the miR-221/miR-222 genomic-locus and initiation of transcription via a long-range interaction between this enhancer and the transcription start site of a primary RNA located ~26kbp upstream of the miR sequences. Reduced levels of miR-221/miR-222 produced by inactivation or deletion of EBNA3A or EBNA3C resulted in an increase in expression of a well-established target of miR-221/miR-222, the cyclin-dependent kinase inhibitor p57KIP2. These data indicate that EBNA3A and EBNA3C contribute to B cell transformation by inhibiting multiple tumour suppressor proteins, not only by direct repression of protein-encoding genes such as BCL2L11 and CDKN2A, but also by the manipulation of host pri-miRs and microRNAs.

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THE ROLE OF INTERACTIONS BETWEEN CELLULAR PROTEINS AND EBV-ENCODED NUCLEAR ANTIGENS IN TRANSFORMATION OF B

LYMPHOCYTES

Elena Kashuba (1, 2), Mariya Yurchenko (2), Surya Pavan Yenamandra (1), Suhas Darekar (1), Boris Snopok (3), Barbro Ehlin-Henriksson (1), Laszlo Szekely

(1) and George Klein (1)

(1) MTC, Karolisnka Institutet, Nobels väg 16, Box 280, Stockholm, Sweden (2) RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology

of NASU, Kyiv, Ukraine (3) VS Lashkarev Institute of Semiconductor Physics of NASU, Kyiv, Ukraine

The fact that EBV can transform normal resting B cells into immortalized lymphoblasts in a single step, through the action of a defined set of viral proteins, makes it an excellent model system to study the molecular mechanisms of cell transformation. Over the last years, we have discovered 17 previously unknown interactions between transforming proteins EBNA-3, EBNA-5, and EBNA-6, and cellular proteins, by using the yeast two-hybrid system and immunoprecipitation followed by mass-spectrometry. The identified proteins are involved in cell metabolism (PHD1, PHD2, HIF1α, UCKL-1), the p53 (p14ARF, MDM2) and RB-E2F (MRPS18-2) pathways, signaling pathways of nuclear receptors (XAP-2, AhR, VDR, HIF1α), and in the control of apoptosis (MDM2, AhR, VDR). We have found that EBNA-6 binds to the mitochondrial ribosomal protein MRPS18-2 and targets it to the nucleus, facilitating its binding to RB. This disrupts the association of E2F1 with RB and promotes the entry into the S-phase. We have found also that EBNA-5 binds to MDM2 and that trimolecular complexes were formed where MDM2 served as a bridge between EBNA-5 and p53. This binding stabilizes the levels of p53 in the EBV infected cells. Tri-molecular complex formation between EBNA-5, MDM2 and p53 inhibits binding of p53 to DNA, and thereby the p53 dependent apoptotic pathway. A Warburg effect, or “aerobic” glycolysis was observed in transformed B cells, in result of HIF1α protein stabilization. This was due to inhibiting of HIF1α protein degradation as a consequence of the binding between prolylhydroxylases PHD1 and PHD2 and EBNA-5 and EBNA-3, respectively.

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Session 3: Pathogenesis

EPSTEIN-BARR VIRUS MIRNAS ATTENUATE THE CELLULAR RESPONSE

TO TYPE I INTERFERON

Wiertz E (1), Hooykaas M (1), van Gent M (1), Boer I (1), Kruse E (1), Willems L (1), van Hooff S (1), Groot-Koerkamp M (1), Holstege F (1), Ressing M (2),

Lebbink RJ (1)

(1) University Medical Center Utrecht, The Netherlands; (2) Leiden University Medical Center, The Netherlands

MicroRNAs are posttranscriptional gene regulators that play important roles in many cellular processes. These short non-coding RNA molecules regulate gene expression by binding to complementary target mRNAs, thereby inducing RNA destabilization and inhibition of translation. Several DNA viruses hijack the cellular miRNA machinery to produce their own miRNAs, which offers opportunities to regulate both cellular and viral gene expression. Epstein-Barr Virus (EBV) encodes more than 40 different miRNAs that are expressed during all stages of the virus lifecycle as well as in EBV-associated tumors. In recent years, the first studies assessing the functions of these miRNAs have been published, although the targets for most EBV miRNAs remain largely unknown. As part of the innate immune system, type I interferons are released upon pathogen detection. They induce an antiviral state and have anti-proliferative effects in target cells. Given the large number of herpesvirus proteins known to interfere with the host immune system, we assessed whether EBV miRNAs could contribute to immune evasion by specifically targeting the type I interferon response. To assess this, we generated a cell line carrying an interferon-stimulated response element (ISRE)-mCherry reporter. EBV miRNAs were expressed in this cell line to study their impact on the reporter. Indeed, upon expression of a large cluster of EBV miRNAs, the response to IFN-α was reduced significantly. Using a panel of custom miRNA inhibitors, we identified EBV miR-BART16-5p as a causative agent for this interferon signalling inhibition. Subsequent gene expression profiling identified the CREB-binding protein (CBP) as potential miRNA target. CBP is a histone acetyltransferase that has been implicated in interferon signalling. 3’UTR reporter assays and western blot analysis confirmed CBP as a direct target for miR-BART16-5p. Our data suggest that EBV miRNAs regulate immune responses by specifically targeting the interferon response, and as such may contribute to EBV immune evasion.

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CELL TYPE SPECIFIC MODULATION OF BCL6 EXPRESSION BY INTERFERONS IN CELL LINES DERIVED FROM HUMAN HEMATOPOIETIC

MALIGNANCIES Salamon D (1), Madapura H (1), Rasul E (1), Nagy N (1), Kallas T (1), Kröhnke M

(1), Ujvari D (1), Björkholm M (2), Klein G (1) and Klein E (1)

(1) Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden; (2) Department of Medicine, Division of Hematology,

Karolinska University Hospital Solna, Stockholm, Sweden BCL6 is the master regulator of the germinal center reaction. It is critically involved in the pathogenesis of several human malignancies. Interferons (IFNs) are multifunctional cytokines with antiviral, antiproliferative, proapoptotic and immunoregulatory properties. Although IFNs have remarkable effects on several hematopoietic malignancies in vitro, they have only limited therapeutic efficiency. This remarkable difference prompted us to study the effects of IFNs on BCL6 expression in human cell lines derived from three hematopoietic malignancies, in which BCL6 was shown to play a role in the pathophysiology of the disease. We found that IFN-gamma is a strong, STAT-1 dependent direct inducer of BCL6 expression in multiple myeloma (MM) and imatinib treated, but not in untreated chronic myeloid leukemia (CML) cell lines. Furthermore, IFN-gamma rescued CML cancer stem cells during imatinib treatment in a BCL6 dependent fashion. In myeloma cells, IFN-alpha only minimally upregulates BCL6, because the concomitant phosphorylation of STAT-5 selectively counteracts the effect of BCL6 inducing IFN-alpha signals. Burkitt`s lymphoma (BL) lines express high level of BCL6 driven by FoxO1. Contrary to MM, where IFN-alpha induces BCL6, in cell lines derived from endemic (but not from sporadic) BL IFN-alpha directly and progressively inhibits BCL6 expression. IFN-alpha induced BCL6 downregulation is independent of Epstein-Barr virus and mediated through the transient activation of STAT-5.

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EBV-INFECTED CELLS IN SLE PATIENTS RELEASE EXOSOMES THAT DELIVER INFLAMMATORY SMALL RNA TO RENAL TUBULAR EPITHELIAL

CELLS

Masoumi N (1), Tsang M (1), Sjoe A (1), van Eijndhoven MAJ (1), Heutinck KM (4), Grunberg K (1), ten Berge RJM (4), Juwana H (1), Riewaerts T (1), Gelderman KA (1), von Blomberg BME (1), Schiffelers RM (5), Vervloet M (3), van de Weering

J (6), Bultink IE (2), Middeldorp JM (1), Voskuyl AE (2) and Pegtel DM(1)

(1) Department of Pathology, Exosomes Research Group, Cancer Center Amsterdam, VU University Medical Center, de Boelelaan 1117 HV Amsterdam,

The Netherlands; (2) Department of Rheumatology, VU University Medical Center, de Boelelaan 1117HV Amsterdam, The Netherlands; (3) Department of

Nephrology VU University Medical Center, de Boelelaan 1117HV Amsterdam, The Netherlands; (4) Department of Internal Medicine, Experimental Immunology and Renal Transplant Unit, Academic Medical Center, Amsterdam, The Netherlands;

(5) Department of Clinical Chemistry, Utrecht University The Netherlands; (6) Department of Cell Biology VU University, The Netherlands

Latent Epstein Barr virus (EBV) infected B cells produce massive amounts of non-coding (nc)RNAs that can be transferred to non-infected target cells altering their physiology. Here we demonstrate that cell-free EBV-ncRNAs present in blood and urine, distinguish Systemic Lupus Erythematosus (SLE) patients from controls. Dissemination of pro-inflammatory EBV-EBER1 small RNA in the blood is mediated via extracellular vesicles (EVs), consistent with exosomal secretion by latent-infected B cells in vitro. High levels of EBER1 were detected in Lupus Nephritis renal biopsies while EBV-DNA was virtually absent. Notably, in situ RNA hybridization reveals accumulation of EBERs in the apical cytoplasm of renal tubular epithelial cells (TECs), indicative of a non-cell autonomous origin. Kidney-injury molecue-1, a phosphatidylserine (PtdSer) surface receptor involved in cellular exosome-capture is expressed in LN tissues and at the cell surface of primary TECs. In analogy to virus uptake, PtdSer mediates internalization of EBER1-carrying exosomes. TEC express multiple RNA sensors that can recognize EBER1 explaining transcription of type-I interferon response genes and IL-6 production upon exosome-mediated EBER1 delivery. Thus in LN, exosomes released by EBV-infected cells can target tubular epithelial cells upon passage of glomeruli and may promote anti-viral inflammatory responses.

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INTEGRIN Α9 GENE PROMOTER IS HYPERMETHYLATED AND DOWNREGULATED IN NASOPHARYNGEAL CARCINOMA

Nawaz I (1,2), Hu LF (1), Du ZM (1,3), Moumad K (4,5), Ignatyev I (1), Pavlova TV

(1), Kashuba V (1), Zabarovsky ER (1,6), Ernberg I (1) (1) Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Box

280, Stockholm SE-17177, Sweden; (2) Department of Microbiology, Faculty of Life Sciences, University of Balochistan, Quetta, Pakistan; (3) State Key

Laboratory of Oncology in South China, and Department of Pathology, Sun Yat-Sen University Cancer Center, Guangzhou, P.R. China; (4) Department of

Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany; (5) Oncovirology Laboratory, Institut Pasteur du

Maroc, 20360 Casablanca, Morocco; (6) Department of Clinical & Experimental Medicine, Division of Cell Biology, Linköping University, Linköping, Sweden

Epigenetic silencing of tumor suppressor genes (TSGs) by promoter methylation can be an early event in the multi-step process of carcinogenesis. Human chromosome 3 contains clusters of TSGs involved in many cancer types including nasopharyngeal carcinoma (NPC), the most common cancer in Southern China. Among ten candidate TSGs identified in chromosome 3 using NotI microarray, ITGA9 and WNT7A could be validated. 5-aza-2’ deoxycytidine treatment restored the expression of ITGA9 and WNT7A in two NPC cell lines. Immunostaining showed strong expression of these genes in the membrane and cytoplasm of adjacent control nasopharyngeal epithelium cells, while they were weakly expressed in NPC tumor cells. ITGA9 promoter was highly differentially hypermethylated whereas WNT7A was only weakly methylated in NPC compared to normal tissues. The expression level of ITGA9 in NPC tumors was downregulated 4.9-fold, compared to the expression in control. ITGA9 methylation was detected by Methylation specific PCR in 56% of EBV positive NPC-cases with 100% specificity. Taken together, this suggests that ITGA9 might be a TSG in NPC that is involved in tumor cell biology. The possibility of using ITGA9 methylation as a marker for early detection of NPC should further be explored.

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EXPRESSION OF CHEMOKINE RECEPTORS CCR1 AND CCR2 FROM THE CHROMOSOME INSTABILITY REGION AT HUMAN 3P21.31 IS IMPAIRED IN

EBV-CARRYING BURKITT’S LYMPHOMA CELL LINES Kholodnyuk Holodnuka I (1), Spaka I (1), Leonciks A (2), Rudevica Z (2), Rivkina A (1,3), Lejniece S (3), Murovska M (1), Ehlin-Henriksson B (4), Imreh S (4), Klein

G (4), Kashuba E (4), Kozireva S (1) (1) A. Kirchenstein Institute of Microbiology and Virology, Riga Stradins University

(RSU), Ratsupites 5, 1067 Riga, Latvia; (2) Latvian Biomedical Research and Study Centre, Ratsupites 1, 1067 Riga, Latvia; (3) Riga East Clinic University Hospital, Chemotherapeutic and Hematological Clinic, Linezera 6, 1006 Riga,

Latvia; (4) Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Nobels väg 16, Box 280, 171 77 Stockholm, Sweden

Earlier we defined a malignancy-related chromosome instability region (CIR) at human 3p21.31. A cluster of the CCR genes, including CCR1, CCR2, CCR3, and CCR5, resides within this CIR. It was reported previously that EBV infection of B-cells leads to the induction of CCR2, CCR6, CCR7, CCR9, and CCR10. Expression of the CCR1, CCR2, and the adjacent genes was analyzed at the mRNA levels in 22 B-cell lymphoma cell lines: 11 EBV-negative and 11 EBV-positive Burkitt’s lymphoma (BL). In EBV-negative cell lines, there was no CCR2 mRNA expression and the CCR1 transcript was only detected in 2. We observed that CCR1 and CCR2 mRNA expression of the exon1/exon2 was induced in 9 and 7, respectively, EBV-positive cell lines. However, the CCR1 and CCR2B Open Reading Frame transcripts were only found in 2 and 4, respectively, EBV-positive LatIII BL cell lines. On the cell surface, CCR1 was detected in two LatIII BL cell lines in a small proportion of the cells (10-12%). There was no CCR2 on the cell surface in any BL cell line. However, we detected both CCR1 and CCR2 on PB circulating monocytes and CD19+CD38+ cells in patients with B-cell lymphomas: FL (n=2), DLBCL (n=2), CLL at III/IV (n=4/40). Immunostaining showed also the presence of CCR1 and CCR2B in healthy donor tonsillar B-cells activated in vitro. Since expression of CCR1 and CCR2 is impaired significantly in BL cell lines, we suggest that CCR1 and CCR2 may have prognostic relevance in lymphomas of activated B-cell type.

22  

THE ROLE OF OXIDATIVE STRESS IN LMP1-INDUCED CHRONIC INFLAMMATION AND CARCINOGENESIS IN VIVO

Gao X, Lampraki EM and Wilson J

College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow,

G12 8QQ, UK Transgenic mice expressing LMP1 in the skin epithelium display a hyperplasia and chronic inflammatory phenotype and are predisposed to carcinogenesis. Carcinogenesis can be enhanced with the use of topical carcinogens. We show that the leukocyte infiltrate in the inflamed tissue has high levels of endogenous reactive oxygen species (ROS). N-acetyl-L-cysteine (NAC), a precursor of glutathione, can scavenge ROS and is thought to be an effective antioxidant in vivo. In order to investigate the effects of antioxidant treatment upon the LMP1 induced phenotype, NAC was supplied in the drinking water of the transgenic and control mice. NAC treatment delayed the age progressive phenotype in LMP1 mice which was quantified though in vivo inflammation imaging. Expression data of proteins involved in inflammation and oxidative stress including YM, TGF-alpha, SOD1, Erk1/2, STAT3 and S100A9 will be presented. NAC treatment also significantly reduced the lesion load in LMP1 transgenic mice treated with chemical carcinogens, however this effect is compounded by body weight loss.

23  

CRISPR/CAS9-MEDIATED GENOME EDITING OF HERPESVIRUSES LIMITS PRODUCTIVE AND LATENT INFECTIONS

Lebbink RJ (1), Van Diemen FR (1), Kruse EM (1), Hooykaas MJG (1), Bruggeling CE (1), Van Ham PM (1), Imhof SM (2), Nijhuis M (1), Wiertz EJHJ (1)

(1) Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands; (2) Department of Ophthalmology, University Medical Center Utrecht,

Utrecht, The Netherlands Herpesviruses infect the majority of the human population and can cause significant morbidity and mortality. Herpesviruses persist in their host for life by establishing a latent infection that is interrupted by periodic reactivation events during which replication occurs. Current antiviral drug treatments target the clinical manifestations of this productive stage, but they are ineffective at eliminating these viruses from the infected host. Here, we set out to combat both productive and latent herpesvirus infections by exploiting the CRISPR/Cas9 system to target viral genetic elements important for virus fitness. To achieve this, we constructed a lentiviral CRISPR/Cas9 vector that allows for stable expression of all CRISPR/Cas9 components in human cells. By targeting gRNAs to essential viral genes, we show effective abrogation of HCMV and HSV-1 replication. Using the same approach, EBV can be almost completely cleared from latently infected EBV-transformed human tumor cells. Our studies indicate that the CRISPR/Cas9 system can be effectively targeted to herpesvirus genomes as a potent prophylactic and therapeutic anti-viral strategy impairing viral replication and clearing latent virus infection.

24  

Key-note lecture

Escape from latency – a proposal

Wolfgang Hammerschmidt

25  

An invited lecturer

Dynamics of EBV-carrying B-cells in a modern representation of Waddingtons landscape

Ingemar Ernberg

26  

Session 4: Immunology

STUDY OF SURFACE ANTIGENIC AND SECRETORY DETERMINANTS OF EBV LATENCY III B CELLS THAT CAN MODULATE THE ANTI-EBV T-CELL

RESPONSE Auclair H (1), Ouk C (1), Al Mohamad H (1), Faumont N (1), Feuillard J (1, 2) and

Jayat-Vignoles C (1)

(1) UMR CNRS 7276 CRIBL, Faculty of Medicine, University of Limoges, France; (2) Hematology laboratory, CHU Dupuytren, Limoges, France

Epstein-Barr gamma-herpes virus (EBV) infects more than 95% of the human adult population. In vitro it infects and transforms primary B cells, leading to lymphoblastoid cell lines called LCLs (latency III proliferation program). In vivo, EBV can be associated with different types of B cell lymphomas. Understanding overall EBV latency III infected B cells impact on T-cell response modulation (direct interaction combined with micro-environment) is important to elucidate EBV-related lymphomagenesis and better define EBV-associated lymphoproliferative disorders. The aim of this study was to better characterize antigenic determinants and micro-environment of EBV latency III B cells in connection with anti-EBV T-cell responses. Analyses were performed on Burkitt lymphoma cell lines infected (BL2B95.8, BL41B95.8) or not (BL2, BL41) by EBV and on LCLs. Expression of antigenic determinants was assessed by flow cytometry. Transcriptional and secretory activities of cytokines were respectively estimated by qRT-PCR and ELISA. Our previous results have shown that EBV infected B cells in latency III can modulate anti-EBV T-cell responses by over-expression of the pro-apoptotic receptor Fas (sensitization to death) on cell surfaces, as well as the immuno-inhibitor B7H1/PDL1 (inhibition of T cell cytotoxicity). To further understand the overall immune-regulatory role of EBV infected B cells on the immune response and consequently lymphomagenesis, we studied expression of several major molecules of the immunological synapse. We observed that LCLs overexpressed CMHI, CMHII and molecules of the B7 family. Additional data on transcriptional and secretory activities of some inflammatory and immuno-suppressive cytokines highlighted that the two immuno-suppressive cytokines IL-10, and at a lower level TGF-beta, were associated with the latency III program. Different profiles that potentially inhibit T cell responses were observed for different LCLs. Altogether, despite overexpression of molecules involved in T-cell killing of EBV infected B-cells such as CD80 or CD86, the balance seems to tip in favor of EBV-regulated expression of T-cell inhibitory molecules.

27  

EBV IMMUNE EVASION OF NK CELL KILLING: VIRUS INFECTED B CELLS ARE PROTECTED AGAINST NK CELL LYSIS DURING LATE STAGE LYTIC

CYCLE

Williams L, Quinn L, Rowe V, Zuo J

School of Cancer Sciences and Centre for Human Virology, the University of Birmingham, B15 2TT, UK

Epstein-Barr Virus (EBV) persists for the lifetime of the infected host despite eliciting strong immune responses. This persistence requires a fine balance between the host immune system and EBV immune evasion. Accumulating evidence suggests an important role for natural killer (NK) cells in this balance. NK cells can kill EBV infected cells undergoing lytic replication in-vitro and studies in both humans and mice with reconstituted human immune systems have shown NK cells can limit EBV replication and prevent infectious mononucleosis. Despite this strong interaction between NK cells and EBV, previous reports have suggested that EBV does not possess any NK cell evasion mechanisms. We now show that NK cells, via NKG2D and DNAM-1 interactions, recognize and kill EBV infected cells undergoing lytic replication, and that expression of a single EBV lytic gene, BZLF1, is sufficient to trigger this killing. Contrary to previous reports we show that EBV possesses at least one NK cell immune evasion strategy. Expression of BHRF1 in BZLF1+ cells protects the cells from NK cell killing. This is not mediated through changes in NK cell ligands but, we hypothesise, through the anti-apoptotic function of BHRF1. We show that this protection is physiologically relevant in that EBV infected cells in late lytic cycle are protected from NK cell killing. This is the first evidence of EBV evading NK cell mediated immunity and further explains how EBV can persist in immunocompetent hosts.

28  

EXOSOMAL-SORTING OF 5’PPP-EBER1 RNA EVADES CYTOSOLIC SENSING IN LATENT EBV-INFECTED B CELLS BUT SUPPORTS

RECOGNITION BY DENDRITIC CELLS

Pegtel DM (1), van Eijndhoven MAJ (1), Koppers-Lalic D (1), Hackenberg M (4), Masoumi N (1), Neville N (1), Lougheed SM (2), Berenguer J (4), Hopmans E(1),

Voskuyl AE (1), Swaminathan S (1), Gibbs S (1), van Rinkel RNPM (2), Greijer AE (1), Verkuijlen S (1), Knol J (1), Scheffer GL (1), van Kuppeveld F (1), Wurdinger T

(5,6), Jordanova K (1), de Gruijl TD (1,2), Jimenez C (8), Baglio SR (1), and Middeldorp JM (1)

(1) Department of Pathology, Cancer Center Amsterdam, VU University Medical

Center, de Boelelaan 1117 HV Amsterdam, The Netherlands; (2) Medical Oncology, VU University Medical Center, de Boelelaan 1117HV Amsterdam, The Netherlands; (4) Genetics Department, University of Granada, 18071 Granada, Spain; (5) Neuro-oncology Research Group, Department of Neurosurgery, VU

University Medical Center, 1081HV Amsterdam, The Netherlands; (6) Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital,

Harvard Medical School, Boston, MA; (8) OncoProteomics Laboratory, Dept. Medical Oncology, Cancer Center Amsterdam, VU University Medical Center, de

Boelelaan 1117HV Amsterdam, The Netherlands The successful herpes virus Epstein Barr (EBV) protects its host from detrimental consequences of infection. Despite massive transcription of pro-inflammatory small RNAs (EBERs) latent EBV escapes antiviral immunity. Here we demonstrate that in absence of the Pol III transcript-binding ribonucleoprotein lupus antigen (La) uncapped 5’triphosphate (5’ppp)EBER1 supports cytosolic sensing. Importantly, inhibition of endosomal small RNA sorting into exosomes triggered IFN-beta transcription in latent EBV-infected B cells. Moreover EBER1 dissociated from La, is selectively exported via exosomes that can target plasmacytoid dendritic cells (pDCs). Using an EBER-deficient viral strain, enzymatic removal of 5’ppp of the exosomal small RNA content and EBER1 in vitro generated transcripts, we established that EBER1-delivery via exosomes drives a type I anti-viral response in target DCs. In vivo, we could detect EBER1, but no EBV-DNA, in pDC-infiltrated, inflamed skin lesions of cutaneous- and chronic discoid lupus erythematosus patients. Thus, latent EBV-transcripts escape cytosolic sensors in infected B cells whereas antiviral immunity is triggered upon exosomal sorting and capture by sensory recipient cells, shedding a new light on the host-pathogen standoff.

29  

THE MISSING LINK IN EBV IMMUNE EVASION: THE BDLF3 GENE INDUCES UBIQUITINATION AND DEGRADATION OF MHC CLASS I AND MHC CLASS II

Quinn L, Williams L, Zuo J, Rowe M

School of Cancer Sciences and Centre for Human Virology, University of Birmingham, B15 2TT, UK

Epstein Barr Virus (EBV) infection triggers CD8 and CD4 T cell responses yet persists for the lifetime of infected hosts. The ability of EBV to persist relies upon a balance between host immune responses and EBV immune evasion. CD8 T cell responses to EBV show a pattern of immunodominance whereby late (L)-antigen-specific T cells are rarely detected compared to immediate-early and early. Moreover, the ability of CD8 T cells to recognise infected cells diminishes as lytic cycle progresses. This is due to the activity of EBV immune evasion proteins including BILF1, which shows strong interference with CD8 T cell recognition during L-lytic cycle. However, protection provided by BILF1 does not explain the ultimate level of protection seen at L-lytic cycle. We have identified the L-gene BDLF3, as the missing link to account for efficient evasion during L-lytic cycle. Furthermore, BDLF3 contributes to the evasion of CD4 T cell responses to EBV, a phenomenon that is well-established but for which molecular mechanisms are lacking. In the present study we demonstrate that BDLF3 expression results in downregulation of surface MHC class I and class II by inducing rapid internalisation and delaying appearance of MHC molecules. Reduced MHC expression correlates with protection against CD8 and CD4 T cell recognition. The molecular basis of surface MHC molecule loss is identified as BDLF3-induced ubiquitination of MHC molecules and subsequent degradation in a proteasomal dependent manner.

30  

A NOVEL VIRAL IMMUNE EVASION MOLECULE INTERFERES WITH ANTIGEN PRESENTATION DURING THE LATE PHASE OF EPSTEIN-BARR

VIRUS INFECTION Gram AM, Lindenbergh M, Geerdink R, Lebbink RJ, Hoeben RC, Wiertz EJ, and

Ressing ME

Dept Molecular Cell Biology, LUMC, Leiden, The Netherlands; Dept Medical Microbiology, UMCU, Utrecht, The Netherlands

Persistent viruses employ ingenious strategies to evade elimination from infected hosts. For instance, EBV interferes with CD8+ T cell immunity early during productive infection. Besides HLA class I (HLA I)-restricted T cells, also natural killer T (NKT) cells contribute to anti-viral host defense when activated by lipids presented by CD1d molecules. Here, we studied if EBV interferes with CD1d-restricted antigen presentation. We found that productive infection with EBV reduced CD1d surface expression on antigen-presenting B cells. We identified BDLF3, encoding an envelope glycoprotein of 150 kDa that had no known function, as contributing to CD1d evasion. To our surprise, cellular expression of the highly glycosylated gp150 also caused downregulation of surface expression of the peptide-presenting HLA I and II molecules. B cells infected with a gp150-deficient virus had a reduced immune evasion phenotype and this was reverted by restoration EBV gp150 expression. Mechanistically, gp150 impeded detection of the three antigen-presenting molecules through glycan-mediated steric shielding. In conclusion, we elucidated a function of EBV gp150, namely interference with antigen presentation by CD1d, HLA I and II. The expression of gp150 late during productive EBV infection, implies that it could contribute to undetected virus production by mediating evasion from (NK)T cell immunity. Glycan-shielding represents a novel (NK)T cell evasion strategy of human herpesviruses.

31  

Session 5: Therapy and disease models

MALIGNANT LYMPHOMA IN PATIENTS BEFORE AND AFTER THE INTRODUCTION OF COMBINATION ANTIRETROVIRAL THERAPY (C-ART)

Grutzmeier S (1,2), Hedberg V (1,2), Svedmyr E (3) and Ernberg I (2)

(1) Department of Venhälsan/Infectious Diseases, South General Hospital; (2)

Department of Microbiology, Tumor and Cell Biology(MTC), Karolinska Institutet, Stockholm; (3) Department of Hematology, Karolinska University Hospital Solna

Background: Malignant lymphoma and Kaposi’s sarcoma were the most common tumors before c-ART in HIV-infected patients. Kaposi’s sarcoma is associated with Human Herpes Virus 8 (HHV-8) reactivation, Lymphoma with Epstein Barr virus (EBV) reactivation and in some cases HHV-8 reactivation. Material and methods: Since the start of the clinic we have followed all patients with HIV-infection from diagnosis of the infection 2-4 times a year. From 1989-1996 we had a special cohort which was investigated for reactivation of CMV-infection and all other opportunistic infections and cancers. All lymphomas was treated according to protocol by ES and followed at our clinic afterwards. Diagnosis of Lymphoma was done by biopsy and also by post mortem in most cases. The periods looked at are from 1982-1996 (before c-ART) and 1997-2012 (after c-ART). Results: From 1982 through 1996 thirty-seven malignant lymphomas was diagnosed, 36 Non-Hodgkin lymphoma (NHL) and one Hodgkin Disease (HD), and from 1997 through 2012 11 NHL and 2 HD. Incidense of lymphoma was 4,5% in the first group and 1% in the second group. Of the 36 NHL in the first period 16 were CNS lymphoma, 14 B-cell-lymphoma, 4 T cell lymphoma, one 0- cell lymphoma and one immunoblastic lymphoma. The two latter caused by HHV-8 reactivation. Of the 16 lymphomas in the second period 11 were B-cell lymphoma and 2 HD. No lymphoma caused by HHV-8 was seen after introduction of c-ART. Survival in the first group was 10% and in the last group 92%. Conclusion. The incidence of Lymphoma has decreased after introduction of c-ART, but is still more than in the background population.CNS-lymphomas and T-cell lymphomas has disappeared since introduction of c-ART and we have seen no HHV-8 related Lymphomas after introduction of c-ART. Prognosis has also improved with a survival comparable to lymphomas in non HIV-infected persons.

32  

EPSTEIN-BARR VIRUS REACTIVATION FOLLOWING STEM CELL TRANSPLANTATION IS ASSOCIATED WITH THE EARLY EMERGENCE OF

CIRCULATING CD27+ MEMORY B CELLS Bell A (1), Burns D (1), Tierney R (1), Shannon-Lowe C (1), Croudace J (1), Inman

C (1), Chaganti S (2), Craddock C (2), Moss P (1,2) and Rowe M (1)

(1) School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom; (2) Centre for Clinical Haematology, Queen Elizabeth Hospital, UHB NHS Foundation

Trust, Edgbaston, Birmingham, B15 2TH, United Kingdom Stem cell transplant patients provide a unique opportunity to track EBV infection in the context of a newly emerging B cell system. While many stem cell recipients maintain low or undetectable EBV loads post-transplant, a significant minority exhibit elevated and rapidly increasing EBV loads which can lead to potentially fatal EBV-associated post-transplant lymphoproliferative disease. Intriguingly this high level EBV reactivation typically arises in the first three months post-transplant, at a time when the reconstituting B cell system is dominated by transitional and naive cells, rather than CD27+ memory cells which are the usual site of EBV persistence. To investigate this apparent paradox, we prospectively monitored EBV levels during B cell reconstitution in a cohort of patients for up to 12 months post-transplant. In patients with low EBV loads, the peripheral blood B cell pool was dominated by CD19+ CD27- IgD+ naïve cells, with relatively few CD19+ CD27+ memory cells. However, patients with high EBV loads had significantly increased numbers of memory B cells, up to 90% of which were latently infected with EBV. Further analysis revealed that many of these CD27+ B cells expressed EBV transcripts associated with growth transformation and were Ki-67 positive. Together, these data suggest that high level EBV reactivation following stem cell transplantation may drive the expansion of latently infected CD27+ B lymphoblasts in the peripheral blood.

33  

THE EFFECTIVENESS OF COMBINED ANTIVIRAL THERAPY IN CHRONIC MONONUCLEOSIS CAUSED BY EPSTEIN-BARR VIRUS

Maltsev DV, Kazmirchuk VE

Institute of Immunology and Allergology, O’Bogomolets National Medical

University (Ukraine) The Epstein Barr virus is a well known etiologic factor of chronic mononucleosis syndrome, which includes fever, pharingitis, lymphadenopathy, splenomegaly, and presence of atypical mononuclear cells in the blood. The aim: to evaluate the effectiveness of the combined antiviral therapy in chronic mononucleosis caused by the Epstein-Barr virus. Materials and methods: the studied group consisted of 72 adult patients with chronic mononucleosis that showed positivity to EBV based on a PCR analysis with specific primers. These patients received valacyclovir at a dose of 3 g/day per os and also recombinant alpha-2b-interferon at a dose of 3 million IU i/m per day. The control group consisted of 63 similar patients who did not pass antiviral treatment. Results: we observed no PCR signal after the analysis of the serum after the first month of treatment in 52% of patients, after 2 months – in 21%, and at the end of 3rd months – additionally in 17% of cases. Resistance to treatment showed only 10% of the patients in the study group. Manifestations of lymphoproliferative syndrome disappeared in 1-3 weeks after the removal of reactivated infections, whereas normalization of body temperature usually coincides with the moment of removal of the virus from the blood serum. After treatment, most patients improved quality of life. In the control group spontaneously normalized PCR results occurred in only 5 patients. Conclusions: Combination of antiviral therapy, i.e. valacyclovir and recombinant alpha-2b-interferon is effective in treatment of chronic mononucleosis caused by EBV.

34  

MODELING EBV LATENCY III DRIVEN TUMORIGENESIS IN MICE

Sommermann T, Yasuda T, Wirtz T, Weber T, Rajewsky K

Max Delbrück Center for Molecular Medicine Berlin-Buch, Berlin, Germany The incidence rate of Epstein Barr Virus (EBV) positive lymphomas is dramatically increased in immune compromised patients, underscoring both the transforming potential of EBV and the crucial role of the immune system in the control of EBV driven lymphomas. The goal of our work is to create transgenic mouse models that recapitulate EBV driven lymphomagenesis and the immune response against such tumors. In a first model we showed that LMP1 expressing B cells are cleared by the murine immune system. In T cell deficient mice LMP1 expressing B cells develop rapidly into lymphomas. We now investigate the mechanism of activation and killing of LMP1 B cell specific by T cells. Further, we improve our model to better mimic real EBV expression patterns. To closer model EBV latency III, we now co express LMP1, LMP2A and EBNA3A. Like LMP1 and LMP2A, EBNA3A retains key functions in mouse B cells. We find that EBNA3A interacts with RBPJkappa, suppresses INK4A/ARF and inhibits apoptosis in naïve, cytokine stimulated as well as LMP1/LMP2A expressing B cells. Interestingly EBNA3A also reduces proliferation and differentiation of such stimulated B cells. In vivo, EBNA3A expression inhibits B cell differentiation into germinal center and B1a B cells, indcating that EBNA3A not only inhibits apoptosis but actively regulates B cell differentiation. Currently we study synergistic effects of LMP1, LMP2A and EBNA3A on mouse B cell physiology and lymphomagenesis.

35  

Key-note lecture

Interactions of viruses with genetic modifications in carcinogenesis – special implications for the Epstein-Barr virus system

Harald zur Hausen

36  

POSTERS

(1) P38 B-CELL STATUS SEEMS TO DETERMINE ABILITY TO ENTER IN EBV REACTIVATION OR APOPTOSIS DURING TREATMENT BY THE HDAC

INHIBITOR VORINOSTAT

Al Mohamad H (1), Auclair H (1), Ouk C (1), Rogez S (1, 2), Feuillard J (1,3) and Jayat-Vignoles C (1)

(1) UMR CNRS 7276 CRIBL, Faculty of Medicine, University of Limoges, France;

(2) Virology laboratory, CHU Dupuytren, Limoges, France; (3) Hematology laboratory, CHU Dupuytren, Limoges, France

EBV gamma-herpesvirus infects more than 95% of the population worldwide. Upon infection the virus establishes a latent infection predominantly in B cells and remains in the host during its lifetime. After latency establishment (latency I, II or III, according to expressed viral latency genes), only a small fraction of the virus undergoes reactivation. Latency as well as lytic switch are essential for EBV survival in the host. However, stimuli that trigger or prevent reactivation are not clear. Different chemical or biological agents, such as HDAC inhibitors (iHDAC), are known to trigger reactivation in vitro and can be used to study latency disruption. This is the purpose of this study. Analyses were performed on Burkitt lymphoma cell lines infected (BL2B95.8, BL41B95.8) or not (BL2, BL41) by EBV. Cell lines in an EBV lytic cycle (B95.8), reactivable (P3HR1, latency I) or LCLs (lymphoblastoid cell lines, latency III) were also used. Cells were treated or not with vorinostat, an iHDAC, classified as an EBV reactivator. We observed that in various cell lines a few cells spontaneously underwent reactivation, independently of the latency program. Otherwise, vorinostat treatment of EBV infected B cells led to different cell line outcomes: reactivation, apoptosis or the two simultaneously, with an apparent mutual inhibition between commitmentto apoptosis or reactivation for a given cell. iHDAC is known to trigger EBV immediate-early gene transcription and reactivation as well as activation of apoptosis, suggesting that cell status is a determining factor in ability to go one way or another. First results suggest that p38 could be a determinant factor since high levels of constitutive p38 phosphorylation were associated with EBV reactivation. This question is of interest since promoting apoptosis during treatment of EBV-associated lymphoproliferative disorders by iHDAC could avoid EBV virion release in patients.

37  

(2) HETEROGENEITY OF THE MAJOR INTERNAL REPEAT (IR1) OF EPSTEIN-BARR VIRUS INCLUDES A NONSENSE MUTATION OF EBNA-LP IN

THE PROTOTYPE STRAIN B95-8

Ba Abdullah M (1), Palermo R (1), Palser A (2), Grayson N (2), Kellam P (2) and Rob White (1)

(1) Section of Virology, Faculty of Medicine, Imperial College London, London, UK

(2) Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK The internal repeat 1 (IR1) of Epstein-Barr virus (EBV) is an approximately 20kb array of 3kb repeat units. IR1 contains: Wp - the promoter that initiates the transformation of infected B-cells into lymphoblastoid cell lines (LCLs); exons encoding the repeat domain of latency protein EBNA- LP; BWRF1 - an open reading frame of unknown function. Here, we have characterised the sequence diversity of IR1 for 73 EBV strains isolated from spontaneous LCLs, primary malignancies and tumour-derived cell lines. We find that EBNA-LP exons, and the short intron between them are highly conserved, with greater sequence diversity across the BWRF1 region. Analysing IR1 heterogeneity within each strain showed that 80% of strains contained heterogeneous repeats. The most frequently heterogeneous positions were also SNPs, suggesting this heterogeneity arose through recombination between strains. One striking heterogeneity outside SNPs, was a previously unreported nonsense mutation in one copy of the W1 exon of the prototype lab strain B95-8, that could potentially produce truncated EBNA-LP. Analysis of BWRF1 is ongoing. Overall, only 20% of EBV strains had homogeneous repeats, but all but one of these were spontaneous LCLs, suggesting that IR1 heterogeneity is more prevalent in EBV in cancers and lab strains than circulating EBV strains.

38  

(3) EPSTEIN-BARR VIRUS ENCODED MICRORNAS TARGET SUMO-REGULATED CELLULAR FUNCTIONS

Callegari C and Masucci MG

Department of Cell and Molecular Biology, Karolinska Institutet, S-171 77

Stockholm, Sweden Post-translational modification by the Small Ubiquitin-like MOdifier (SUMO) regulates the cellular response to different types of stress and plays a pivotal role in the control of oncogenic viral infections. Here we have investigated the capacity of microRNAs (miRNAs) encoded by Epstein-Barr virus (EBV) to interfere with the SUMO signaling network. Using a computational strategy that scores different properties of miRNA-mRNA target pairs, we have identified a minimal set of 575 members of the SUMO interactome that may be targeted by one or more EBV miRNA. A significant proportion of the candidates cluster in a functional network that controls chromatin organization, stress and immune responses, DNA damage responses, apoptosis, and TGF-beta signaling. Multiple components of the TGF-beta signaling pathway were inhibited upon upregulation of the BHRF1 encoded miRNAs in cells transduced with recombinant lentiviruses or entering the productive virus cycle. These findings point to the capacity of viral microRNAs to interfere with SUMO-regulated cellular functions that control key aspects of viral replication and pathogenesis.

39  

(4) MAINTENANCE OF TELOMERE HOMEOSTASIS IN EBV INFECTED B-LYMPHOCYTES

Chen X, Kamranvar SA and Masucci MG

Department of Cell and Molecular Biology, Karolinska Insitutet, Stockholm,

Sweden Telomeres are nucleoprotein structures that protect the ends of linear chromosomes. They are shortened at each cell division because of incomplete replication of the chromosome termini. When the length of telomeres falls below a critical threshold, p53 is activated and induces p16- and retinoblastoma protein-dependent replicative senescence. Tumor cells escape this proliferation barrier by two alternative mechanisms, a common one dependent on de novo synthesis of telomeric DNA by telomerase, and a rare one based on telomere recombination known as alternative lengthening of telomeres (ALT). In ALT-positive cells, telomere elongation is mainly accomplished by homologous recombination (HR) of telomeric sequences. This process is repressed by telomere coating shelterin protein complex and the Ku70/80 heterodimer in telomerase-positive cells The evasion of proliferative senescence is a key event in viral oncogenesis. Here we show that newly EBV-infected cells exhibit multiple signs of telomere dysfunction, including the occurrence of extra-chromosomal telomeres, telomere fusion and telomere length heterogeneity, and undergo progressive increase in telomere length without a parallel increase in telomerase activity. This phenotype is accompanied by the accumulation of telomere-associated promyelocytic leukemia nuclear bodies (APB) and telomere sister chromatid exchange (T-SCE), suggesting that EBV infection promotes the activation of ALT. Newly infected cells also display a significant reduction of telomere-associated protein TRF2 and express low levels of TRF1, TRF2, POT1 and ATRX, pointing to telomere de-protection as an important correlate of ALT activation. Collectively, these findings highlight the involvement of recombination-dependent mechanisms for maintenance of telomere homeostasis in EBV-induced B-cell immortalization.

40  

(5) DOES THE HOST MICROBIOTA INFLUENCE EBV INFECTION?

Grasso F (1), Peucker M (1), Johansson A (2), Frisan T (1), Masucci MG (1)

(1) Department of Cell and Molecular Biology, Karolinska Institutet; (2) School of Dentistry Molecular Periodontology, Umeå University

Epstein–Barr (EBV) is a human virus that infects more than the 90% of the population. The virus does not cause major symptoms in the majority of infected people, but it’s associated with several lymphoid and epithelial malignancies. Although epithelial tumors represent 80% of all EBV-related malignancies, very little is known regarding the mechanisms of infection, the establishment of latent versus lytic infection, and the transformation events in epithelial cells. One feature of EBV-related epithelial malignancies, such as nasopharyngeal carcinoma and EBV-associated gastric cancers, is their location in body cavities colonized by the host microbiota. We hypothesize that the interplay between bacteria and EBV in such a milieu influences the efficiency of infection, the establishment of infection, promotes an alteration of the equilibrium between latent and lytic infection, and possibly cell transformation. To test this hypothesis, we are studying the effects of bacterial infection on two EBV positive cell lines of epithelial and lymphoid origin, AGS Bx1 and Akata Bx1, respectively. Preliminary data suggest that bacteria are able to activate the lytic cycle in both cell lines. This effect requires viable bacteria and is mediated by a secreted bacterial effector(s). Further analysis is ongoing to determine the fate of the cells in conditions that mimic a long-term interaction with bacteria.

41  

(6) INTERSTITIAL LUNG DISEASE AND EPSTEIN-BARR VIRUS INFECTION

Kazmirchuk V

Institute of Immunology and Allergology at the Bogomolets National Medical University, Kyiv, Ukraine

A number of patients with interstitial lung lesions with active forms of EBV infection were increased in the last decade, and also in Ukraine. Patients complain on a malaise, chest tightness and dyspnea in varying degrees of severity that are not of cardiac genesis. Percussion noted the blunting of sound in the lower lung sections. The pathological changes, such as fibrosis without changing the structure of the lung roots was detected, using the X-ray on the chest. In rare cases, these changes were combined with an enlargement of the mediastinum. These patients were diagnosed with different pathologies - chronic obstructive pulmonary disease, pneumonia, tuberculosis, and lung cancer. In 92% of these cases an active replication of EBV was confirmed, sometimes in conjunction with HHV-6. Number of EBV DNA determined in saliva was 12x102 copies / mL. In 34% of cases EBV was found in the blood serum. The diagnosis of interstitial pneumonitis in patients who were followed for at least 6 months was set on the basis of clinical and radiological findings in the lungs. As a case report, a child of 5 years was described with interstitial disease of lungs, kidneys, eye and salivary glands. We proposed a mechanism of formation of granulomatous lung disease back in 2003, and hypothesized that EBV might play a role in its formation. Among the observed patients we faced the frequent use of antibiotics, non-steroidal anti-inflammatory drugs, and antihistamines. In our opinion, the foregoing factors including stress and psycho-emotional overload contributed to a decrease in the number of NK cells that led to the development of pathologies associated with lymphotropic herpes virus infections by lowering the immune resistance to these pathogens.

42  

(7) EBV-ENCODED MICRORNAS REGULATE THE SUMOYLATION BY TARGETING RNF4

Jinlin Li (1), Stefano Gastaldello (2), Simone Callegari (1), Omid Faridani (3) and

Maria Masucci (1)

(1) Department of Cell and Molecular Biology, Stockholm, Sweden; (2) Department of Physiology and Pharmacology, Stockholm, Sweden; (3) Ludwig

Institute for Cancer Research, Stockholm, Sweden Covalent attachment of Small Ubiquitin-like MOdifiers (SUMOylation) is a key posttranslational modification of that is widely utilized by virus to benefit their survival and propagation. We have observed a dramatic accumulation of SUMO-conjugates in cell productively infected with Epstein-Barr virus. Thus, triggering of the productive cycle is accompanied by up-regulation of several components of SUMO-conjugation machinery, and by down-regulation of the SUMO-dependent ubiquitin ligase RNF4¬¬. Using a bioinformatics strategy, we predicted that RNF4 may be targeted by the viral microRNA BHRF1-1 that is markedly up-regulated upon during productive infection. This possibility was confirmed in 3’UTR-luciferase reporter assays and further supported by mutation of the miRNA seed site. These results provide novel insight into interaction of EBV and SUMOylation machinery.

43  

(8) MICROENVIRONMENTAL CONTROL ON GROWTH OF BURKITT LYMPHOMAS

Muhammad Mushtaq, George Klein and Elena Kashuba

MTC, Karolisnka Institutet, Nobels väg 16, Box 280, Stockholm, Sweden

 

EBV is associated with 98% of endemic Burkitt lymphomas, but is only present in about of 20% of the sporadic cases. In vivo, infectious virus matures in the keratinizing cells of the pharyngeal epithelium and EBV is shed in saliva then. Oral hairy leukoplakia, frequently observed in AIDS and other immunosuppressed patients, is a microscopically visible focus of productive EBV infection. There is a possibility that any infiltrating mucosa CD21 positive B-cell might be infected by virus directly and fibroblasts, most probably, will be infected with accident lytic cycle induction. There are the epidemiological data that endemic BL (almost 100% EBV positive) starts often as a tumor of jaws or other facial bones, while in USA lymphoma usually starts in abdomen. It was shown before that LPS-activated B-cells and lymphoblasts bind to fibroblasts with a higher affinity, compared with mitogen-activated T-cells. It was shown that the early antigen EA present on NPC cells co-cultivated with EBV-infected B-cells. We started investigation on the interplay between EBV infected B-cells and fibroblasts (epithelial cells as well) in relation to the genesis of nasopharyngeal carcinoma and EBV carrying T cell lymphoma, using the interactive in vitro systems. Preliminary results indicate that fibroblasts grow normally in the presence of mitogen-activated B cells but are killed by EBV infected normal B cells and latency Type I and III BL cells. EBV negative BL cells have no effect.

44  

(9) CYTOKINES PRODUCED BY CD4+T CELLS MODULATE EBV LATENCY TYPES

Nagy N, Salamon D, Heuts F, Rasul E, Madapura H, Rottenberg M, Klein G,

Klein E Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet,

Nobelsväg 16, 17177 Stockholm, Sweden Following infection, EBV is carried for lifetime in memory B cells in a silent state (latency I/0). These cells do not resemble the proliferating lymphoblastoid cells (LCL, latency III) generated after infection. We studied the role of CD4+T cells in this change, in the establishment of EBV latency in B cells. Co-culture of LCLs with activated CD4+T cells led to changes in EBV protein expression pattern. Q-PCR confirmed a shift from latency III towards latency IIa: Cp activity was downregulated, while LMP1 mRNA expression was elevated. This effect was mediated by soluble factors: IL21 and soluble CD40L. In addition, the latency modifying factors were also induced when PBMCs were infected with EBV, as they were able to downregulate Cp promoter in freshly infected B cells physically separated from them (in transwell). However, cord blood derived lymhocytes did not downregulate EBNA2 expression in a similar experiment. To study the in vivo establishment of different EBV latency types, we used humanized mice (NOD/SCID/IL2R-/- mice engrafted with human CD34+ HSC). Q-PCR analysis of viral promoters showed that the relative usage of the viral promoter specific for latency I and IIa (Q promoter) was higher in mice having only CD4+ T cells (CD8+ cells were depleted) and it was absent in CD4+ cell depleted mice. The results showed that CD4+ T cells are responsible for the generation and probably for the growth of latency type I or IIa cells. We emphasize the role of CD4+ T cells in controlling EBV-induced B cell growth by modifying the expression of EBV-encoded proteins.

45  

(10) DETECTION OF NASOPHARYNGEAL CARCINOMA IN MOROCCO (NORTH AFRICA) USING A MULTIPLEX METHYLATION SPECIFIC PCR

BIOMARKER ASSAY

Nawaz I (1,2), Moumad K (3,4), Martorelli D (5), Ennaji MM (5), Zhou X (1,6), Zhang Z (6), Dolcetti R (8), Khyatti M (4), Ernberg I (1), Hu LF (1)

(1) Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Box

280, Stockholm SE-17177, Sweden; (2) Department of Microbiology, Faculty of Life Sciences, University of Balochistan, Quetta, Pakistan; (3) Department of Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany; (4) Oncovirology Laboratory, Institut Pasteur du

Maroc, 20360 Casablanca, Morocco; (5) University Hassan II, Faculty of Sciences and Techniques, Mohammedia - Casablanca, Laboratory of Virology, Microbiology

and Quality/ETB, Mohammedia BP 146, (20650), Morocco; (6) National Cancer Institute - Aviano Medical Oncology Department Cancer Bio-Immunotherapy Unit via F. Gallini, 233081 Aviano (PN) Italy; (7) Department of Orolaryngology – Head

and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, P. R. China; (8) Cancer Bio-Immunotherapy Unit Centro di Riferimento Oncologico

IRCCS - National Cancer Institute Via Franco Gallini 233081, Aviano (PN), Italy Silencing of tumor suppressor genes (TSGs) or activation of oncogenes by e.g. aberrant promoter methylation may be early events during carcinogenesis. The methylation status of such genes can be used for early detection of cancer. We are pursuing this approach in our efforts to develop markers for early detection and follow-up of nasopharyngeal carcinoma (NPC). By multiplex methylation specific PCR (MMSP) multiple relevant genes can be detected simultaneously with high sensitivity and specificity. We have initially screened for 12 potential marker genes, including EBV- genes coding for EBNA1 and LMP1 and ten potential TSGs obtained from previously published data. The resulting assay included EBNA1, LMP1 and three cellular TSGs: ITGA9, RASSF1A and P16. We evaluated this assay on 64 NPC patient biopsies from Morocco, Italy and China compared to DNA from 20 nasopharyngeal control tissues. In the Moroccan NPC-cohort (n=44), prevalence of the EBNA1-gene showed the highest sensitivity (36/44; 82%) with 94% specificity. Out of eight (18%) EBNA1 negative Moroccan samples, only three were positive for at least one methylated cellular gene. By detection of cellular marker genes the sensitivity increased from 82% to 89% (39/44). In the whole material of 64 biopsies from three geographical locations, at least any one marker (viral or cellular) could be detected in 91% biopsies with 90% specificity. In a pilot evaluating assay performance on serum DNA from NPC and controls including samples from Italy (n=11) and China (n=5), at least any one marker from MMSP assay could be detected in 88%, but the specificity was only 50%. An MMSP assay has the potential for detection of NPC by screening in high-risk populations, but saliva seems a better DNA source than sera in order to reach acceptable performance.

46  

(11) THE EPSTEIN-BARR VIRUS NUCLEAR ANTIGEN 1 – A LINK BETWEEN CHROMATIN ORGANIZATION AND GENOMIC INSTABILITY IN VIRAL

ONCOGENESIS

Popov S, Callegari S and Masucci MG

Department of Cell and Molecular Biology, Karolinska Insitutet, Stockholm, Sweden

A rough estimate implicates virus infection in the pathogenesis of up to 20% of all human cancers. Epstein-Barr virus (EBV) is implicated in the pathogenesis of epithelial and lymphoid malignancies such as Burkitt’s lymphoma, Hodgkin disease, NK/T cell lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and post-transplant lymphoproliferative disease, amounting to 1-2% of all tumors globally. The EBV nuclear antigen-1 (EBNA1) is the only viral protein regularly detected in all EBV-associated malignancies and is likely to play a key role in oncogenesis. EBNA1 expression induces oxidative stress and DNA damage. The DNA-damaging effect of EBNA1 has been linked to transactivation of catalytic unit of the NADPH oxidase NOX2. The chromatin organization-altering effect of EBNA1 is mediated by a Gly-Arg rich domain (GR) of EBNA1. Based on the observations that EBNA1 binds to cellular DNA, promotes a genome-wide reorganization of cellular chromatin and alters cellular gene expression we hypothesize that EBNA1 may act as a chromatin remodeler that reprograms the infected cells and cooperates with cellular and viral oncogenes in promoting malignant transformation and that this effect can be mediated by the GR domain. Chromatin immunoprecipitation studies and luciferase assays revealed that EBNA1 binds to the NOX2 promoter. The GR domain is sufficient for binding to and activation of the NOX2 promoter. The results suggest that the GR may act as a “pioneer” factor that remodels chromatin and allows the recruitment of other transcription factors.

47  

(12) CHRONIC LYMPHOCYTIC LEUKEMIA DERIVED PAIRED LINES REPRESENT TWO CLL SUBCLONES IN DIFFERENT STAGES OF

PROGRESSION TOWARDS PROLYMPHOCYTIC LEUKEMIA

Rasul E, Salamon D, Nagy N, and Klein E

Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institute

The EBV carrying paired lines, MEC1 and MEC2, were established earlier from explants of blood derived cells of a CLL patient at different stages of progression to prolymphocytoid transformation (PLL). The lines are unique as they are of same clonal origin and their in vitro proliferation is driven by the same indigenous EBV strain. However, the lines are phenotypically different and represent subsequent sub-clones emerging in the CLL population reflecting the clinical progression of the disease. Proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance. The MEC1line arose before the aggressive clinical stage from an EBV carrying cell within the sub--clone which was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent sub-clone with overt PLL characteristics. The EBV induced proliferation of the MEC cells belonging to the sub-clones with markers of PLL agrees with earlier reports in which PLL cells were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins, EBNA-2 and LMP-1, can be determined at the event of infection as in vitro infected CLL cells express only EBNAs, resulting in non-proliferating cells. These paired lines are particularly important as they provide in vitro cells that represent the sub-clonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells

48  

(13) EPSTEIN-BAAR VIRUS REATIVATION FOLLOWING ALLOGENEIC STEM CELL TRANSPLANTATION IN EARLY PERIOD POST-TRANSPLANT

Trociukas I (1, 2), Chapenko S (2), Murovska M (2), Lejniece S (2, 3)

(1) Riga East Clinical University Hospital, Chemotherapy and Haematology Clinic,

Latvia, (2) A. Kirchenstein Institute of Microbiology and Virology, Rīga Stradiņš University, Latvia, (3) Rīga Stradiņš University, Latvia

Epstein-Barr virus (EBV) reactivation is a frequent event after allogeneic stem cell transplantation (ASCT) that may progress to life-threatening EBV-lymphoproliferative disease (EBV-LPD). Regular monitoring of EBV load in peripheral blood samples after ASCT has helped early identification of high-risk patients and diagnosis of lymphoproliferations. This work aimed to estimate the activity EBV infection in the patients after alogeneic stem cell transplantation. Methods: We followed 35 patients (pts) during 100 days period after ASCT treated with an HLA-genoidentical sibling (n=9; 25.3%) and HLA-matched unrelated donors (n=26; 74.3%). EBV DNA load was quantified in cell-free plasma and in peripheral blood mononuclear cells (PBMCs). For monitoring of EBV load quantitative real-time PCR once a week was performed. Results: Early EBV reactivation (within 100 days after ASCT) developed in 6 pts (17.14%) after HLA-matched unrelated donor ASCT. EBV reactivation was observed in these patients, mostly from 8-12 weeks after transplantation (500 – 305 000 copies/ml). Clinically it was observed fever (6 pts), joint pain, and headache (2 pts). None of the patients after ASCT HLA-genoidentical sibling EBV reactivation in the early period after transplantation was observed. 4 patients with EBV reactivation had received the treatment with a single dose of rituximab (375 mg/m2), but 2 patients – two doses of rituximab. The median time to achieve a negative EBV PCR was 7 days (range 5-21 days). These 6 patients before transplantation was done with T cell depletion with anti-thymocyte globulin (ATG) 2.5 mg /kg (total dose 5 mg /kg) Conclusion: In patients after ASCT from HLA-matched unrelated donors and who received ATG, EBV reactivation was observed more often and treatment with rituximab was effective. Administration of rituximab was well tolerated and appeared to be effective for prevention of EBV-associated LPD.

49  

(14) The frequency and activity of the Epstein-Barr virus infection in patients with immunoglobulin E deficiency

Tsaryk V

Bogomolets National Medical University, Department of Clinical Immunology and

Allergology, Kyiv, Ukraine Isolated deficiency of IgE is the one of the most common primary immunodeficiency, which is still underestimated cause of the health disorders in the modern society. According to our records there are currently no reviews on the issue of isolated IgE-deficiency in humans. That is why it is important to do research in this area. There are no well-established international criteria for the diagnosis of isolated IgE-deficiency at the moment. The aim of our study was to determine the activity of the replication of Epstein-Barr (EBV) virus in biological fluids (blood and saliva), and to identify replicative activity of other lymphotropic viruses, including HHV-6 and CMV in patients with deficiency of immunoglobulin E (<5 IU/ml). We examined 82 patients with IgE-deficiency for the activity and viral replication of EBV, HHV-6, and CMV in saliva and blood, using quantitative PCR. The levels of the total immunoglobulin E in the serum were determined by ELISA. All patients were excluded from primary and secondary immunodeficiencies. Clinically, the patients were noted for refractory chronic rhinosinusitis. We have found an active replication of EBV in 37% of patients with the viral load in saliva 11.1 ± 1.26·102 copies/ml. The active replication of EBV in the blood was found only in 14% of patients. 38% of patients showed the HHV-6 load 9.19 ± 2.13·102

copies/ml, that was considered as the active replication. Active CMV replication was determined only in 2 patients. For comparison, active EBV replication was found only in 21% of patients with other pathology - without IgE-deficiency; the average EBV viral load was 5.62±1.14·102 copies/ml. The active replication of HHV-6 was detected in 32% of patients. Concluding, patients with IgE-deficiency and chronic rhinosinusitis possessed more active forms of EBV and HHV-6, with the greater viral load, in comparison with patients without IgE-deficiency.

50  

(15) PROTEOGLYCAN EXPRESSION CORRELATES WITH THE PHENOTYPE OF MALIGNANT AND NON-MALIGNANT EBV-POSITIVE B-CELL LINES

Tsidulko AY (1, 2), Matskova L (2), Astakhova LA (3), Ernberg I (2),

Grigorieva EV (1, 2) (1) Institute of Molecular Biology and Biophysics SD RAMS, Novosibirsk, Russia; (2) MTC, Karolinska Institute, Stockholm, Sweden; (3) Kemerovo Institute of Food

Science and Technology, Kemerovo, Russia Epstein-Barr virus infection contributes to development of Burkitt’s lymphoma (BL). In this study, a possible involvement of proteogycans (PGs) in EBV - host cell interactions was investigated. To do so, PGs expression was studied in primary B cells and EBV+ cell lines of different phenotypes (lymphoblastoid cell lines (LCLs) and BL cell lines). According to RT-PCR analysis, primary B-lymphocytes expressed different PGs (mainly serglycin, CD44, perlecan, and syndecan-1). EBV+ B cell lines showed lower levels of PGs, moreover, pattern of PG expression was changed. BL cells also showed significant down-regulation of PGs compared to primary B cells. Noteworthy, serglycin was expressed in EBV latency III BL-cells, in contrast to LCLs, while in EBV latency I BL cells both serglycin and perlecan were down-regulated. There was no significant change in PG expression upon 5-AzaC or TSA treatments in LCLs, nor in EBV latency III BL cells- At the same time, several PGs showed up-regulation in EBV latency I BL cells, suggesting partial regulation by epigenetic mechanisms. Similar trends were observed for the key ECM components (collagen 1A1, fibronectin, and elastin). Thus, PGs are expressed in primary B lymphocytes whereas they are not or only partly expressed in EBV-carrying cell lines, depending on their latency program. Expression of PGs in latency I BL cells is silenced due to hypermethylation, but the other mechanism regulates their expression in latency III BL cells. The obtained results show that PG expression patterns are dependent on the EBV latency programs.

51  

List of the participants

Name Affiliation E-mail Abstract on page

Allday Martin Imperial College, London, UK

m.allday@imperial.ac.uk

Al Mouhammad Hazar

University of Limoges, FR

hazar.almouhammad@hotmail.com

36

Auclair Héloïse University of Limoges, FR

auclairheloise@gmail.com 26

Avila Javier CMB KI, Stockholm, SE

Javier.Avila-Carino@ki.se

Ba Abdullah Mohammed

Imperial College London, UK

m.ba-abdullah13@imperial.ac.uk 37

Bell Andrew School of Cancer Sciences, University of Birmingham, UK

A.I.BELL@bham.ac.uk 32

Bernasconi Michele

University Children's Hospital, CH

Michele.Bernasconi@kispi.uzh.ch

Bozoky Benedek KI, Stockholm, SE benedek.bozoky@ki.se Bazot Quentin Imperial College,

London, UK q.bazot@imperial.ac.uk 15

Callegari Simone

CMB KI, Stockholm, SE

simone.callegari@ki.se 38

Carbone Ennio University of Catanzaro, IL MTC KI, Stockholm, SE

Ennio.Carbone@ki.se

Chen Xinsong CMB KI, Stockholm, SE

xinsong.chen@ki.se 39

Correia Paulo Section of Virology Imperial College Faculty of Medicine, London, UK

p.correia12@imperial.ac.uk

Correia Samantha

Section of Virology Imperial College Faculty of Medicine, London, UK

s.correia12@imperial.ac.uk

Ehlin-Henriksson Barbro

MTC KI, Stockholm, SE

Barbro.Ehlin-Henriksson@ki.se

Ernberg Ingemar MTC KI, Stockholm, SE

Ingemar.Ernberg@ki.se

Farrell Paul Section of Virology Imperial College Faculty of Medicine, London, UK

p.farrell@imperial.ac.uk 7

Fitzsimmons Leah

School of Cancer Sciences, University of Birmingham, UK

L.Fitzsimmons@bham.ac.uk 11

Forrest Calum School of Cancer Sciences, University of Birmingham, UK

CXF264@student.bham.ac.uk

Frisan Teresa CMB KI, Stockholm, Teresa.Frisan@ki.se

52  

SE Gao Xiao

College of Medicine, Veterinary and Life Sciences, University of Glasgow, UK

x.gao.2@research.gla.ac.uk 22

Gram Anna

Dept Molecular Cell Biology, LUMC, Leiden

A.Gram@umcutrecht.nl 30

Grasso Francesca

CMB KI, Stockholm, SE

Francesca.Grasso@ki.se 40

Gruffat Henri Centre International de Recherche en Infectiologie -CIRI INSERM, Lyon, FR

hgruffat@ens-lyon.fr 9

Grutzmeier Sven MTC KI, Stockholm, SE

Sven.Grutzmeier@ki.se 31

Gunnell Andrea School of Life Sciences, University of Sussex, Falmer, Brighton, UK

A.Gunnell@sussex.ac.uk

Gupta Soham CMB KI, Stockholm, SE

Soham.Gupta@ki.se

Göndör Anita MTC KI, Stockholm, SE

Anita.Gondor@ki.se

Hammerschmidt Wolfgang

Helmholtz center, Munich, DE

hammerschmidt@helmholtz-muenchen.de

Hu Lifu MTC KI, Stockholm, SE

Lifu.Hu@ki.se 45

Hummler Kerstin CMB KI, Stockholm, SE

kerstin.hummler@gmx.net

Jayat-Vignoles Chantal

University of Limoges, FR

chantal.vignoles@unilim.fr

Kashuba Elena MTC KI, Stockholm, SE

Elena.Kashuba@ki.se 16

Kazmirchuk Vera

Institute of Immunology and Allergology, Bohomolets Medical University, Kyiv, UA

institut_immunologii@ukr.net 41

Kholodnyuk Holodnuka Irina

A. Kirchenstein Institute of Microbiology and Virology, Rīga Stradiņš University, Latvia

irina.holodnuka@rsu.lv 21

Kieff Elliott Harvard University, USA

ekieff@rics.bwh.harvard.edu

Kinch Amelie Infektionskliniken, Akademiska sjukhuset, Uppsala, SE

amelie.kinch@akademiska.se

Klein Eva MTC KI, Stockholm, SE

Eva.Klein@ki.se

Klein George MTC KI, Stockholm, Georg.Klein@ki.se

53  

SE Lebbink Robert Jan

University Medical Center Utrecht, NL

R.J.Lebbink-2@umcutrecht.nl 23

Li Jinlin CMB KI, Stockholm, SE

Jinlin.Li@ki.se

Ljungman Per Center for Allogeneic Stem Cell Transplantation, KUH

Per.Ljungman@ki.se

Maltsev Dmitii Institute of Immunology and Allergology, Bohomolets Medical University, Kyiv, UA

dmaltsev@ukr.net 33

Manet Evelyne INSERM , Lyon University, FR

evelyne.manet@ens-lyon.fr

Masoumi Niala VU University Medical Center, Amsterdam, NL

n.masoumi@vumc.nl 19

Matskova Liudmila.

MTC KI, Stockholm, SE

liudmila.matskova@ki.se 12

Minarovits Janos

University of Szeged minimicrobi@hotmail.com

Moridi Thomas MTC KI, Stockholm, SE

thomas.moridi@stud.ki.se

Mushtaq Muhammad

MTC KI, Stockholm, SE

Muhammad.Mushtaq@ki.se 43

Nagy Noemi MTC KI, Stockholm, SE

Noemi.Nagy@ki.se 44

Nawaz Imran MTC KI, Stockholm, SE

imran.nawaz@ki.se 45

Osborn Kay University of Sussex, Brighton, UK

ko54@sussex.ac.uk 13

Palermo Richard Imperial College London, UK

r.palermo@imperial.ac.uk

Pegtel Michiel VU University Medical Center, Amsterdam, NL

d.pegtel@vumc.nl 28

Popov Sergej CMB KI, Stockholm, SE

sergej.popov@ki.se 46

Quinn Laura School of Cancer Sciences, University of Birmingham, UK

L.L.Quinn@bham.ac.uk 29

Rasul Eahsan MTC KI, Stockholm, SE

eahsan.rasul@ki.se 47

Salamon Daniel MTC KI, Stockholm, SE

Daniel.Salamon@ki.se 18

Shannon-Lowe Claire

School of Cancer Sciences, University of Birmingham, UK

C.ShannonLowe@bham.ac.uk

Sinclair Alison J University of Sussex, Brighton, UK

A.j.sinclair@sussex.ac.uk

54  

Sommermann Thomas

Max-Delbrück-Center for Molecular Medicine, Berlin, DE

thomas.sommermann@mdc-berlin.de

34

Stip Maria MTC KI, Stockholm, SE

marjoleinstip@gmail.com

Styles Christine Imperial College, London, UK

christine.styles10@imperial.ac.uk

Szymula Agnieszka

Imperial College London, UK

a.szymula12@imperial.ac.uk 10

Trociukas Ilze Riga East Clinical University Hospital, Latvia

ilze.trociukas@gmail.com 48

Tsaryk Vladislav Institute of Immunology and Allergology, Bohomolets Medical University, Kyiv, UA

tsarykv@me.com 49

Tsidulko Anna Institute of Molecular Biology and Biophysics SD RAMS, Novosibirsk, Russia

50

Vinas Gaza Helena

MTC KI, Stockholm, SE

helena.vinas.gaza@ki.se

Wahlgren Mats MTC KI, Stockholm, SE

Mats.Wahlgren@ki.se

van Domselaar Robert

CMB KI, Stockholm, SE

robert.van.domselaar@ki.se

West Michelle School of Life Sciences, University of Sussex, Falmer, Brighton, UK

m.j.west@sussex.ac.uk

White Rob Imperial College London, UK

robert.e.white@imperial.ac.uk

Wiertz Emmanuel

University Medical Center Utrecht, NL

ewiertz@umcutrecht.nl 17

Williams Luke School of Cancer Sciences, University of Birmingham, UK

L.R.Williams@bham.ac.uk 27

Winberg Gösta CMB KI, Stockholm, SE

Gosta.Winderg@ki.se

Wood David School of Life Sciences, University of Sussex, Falmer, Brighton, UK

C.D.Wood@sussex.ac.uk 8

Zou Jiezhi MTC KI, Stockholm, SE

Jiezhi.Zou@ki.se

zur Hausen Harald

Deutsches Krebsforschungszentrum, Heidelberg, GE

zurhausen@Dkfz-Heidelberg.de