5 th <strong>European</strong> <strong>Congress</strong> <strong>of</strong> <strong>Virology</strong>Saturday 14 th September 2013, 14h45 – 16h45WORKSHOP 1: “ONCOGENIC MECHANISMS OFVIRUSES”Chairpersons: Massimo TOMMASINO (Lyon, FRANCE) &Fabien ZOULIM (Lyon, FRANCE)Room Tête d’OrKEYNOTE:How does Kaposi Sarcoma-associated Herpes virus cause a vasculartumour?Thomas F. SCHULZInstitute <strong>of</strong> <strong>Virology</strong>, Hannover Medical School, Hannover, GERMANYKaposi Sarcoma-associated Herpesvirus (KSHV), aka HHV 8, is a classI carcinogen as defined by the International Agency for Research againstCancer (IARC/WHO). This classification is based on epidemiological findingsas well as laboratory data suggesting that KSHV, or some <strong>of</strong> itsproteins and miRNAs, have oncogenic properties in experimental settings.However, the risk for a KSHV-infected individual to develop Kaposi Sarcoma(KS) is low (incidence <strong>of</strong> classic KS is 1 -3/100 000 <strong>of</strong> a populationwith a KSHV seroprevalence <strong>of</strong> 20-30%), unless additional conditions,such as immune suppression or inflammation, are present (> 1000 foldexcess risk <strong>of</strong> KS in HIV-infection and approx. 200 fold excess risk intransplant recipients). KSHV thus differs markedly from some humanpapillomaviruses (risk <strong>of</strong> a persistent HPV-16 infection to lead to cervicalcancer in the 10-15% range) or Human T-cell tropic virus I (life time risk<strong>of</strong> an infected individual to develop Adult T-cell leukaemia in the range<strong>of</strong> 1 – 3%). These epidemiological data suggest the need for a carefulinterpretation <strong>of</strong> experimental findings that suggest the rapid appearance<strong>of</strong> tumours in animal models <strong>of</strong> KS.On the other hand, the strong accelerating role <strong>of</strong> immune suppression andinflammation in the pathogenesis <strong>of</strong> Kaposi Sarcoma suggest that KSHVprovides an interesting model to study the interplay <strong>of</strong> inflammation anda carcinogenic virus. In this talk I will review experimental findings thatpoint to a role <strong>of</strong> KSHV in the aberrant angiogenesis seen in KS lesionsand the role <strong>of</strong> inflammatory stimuli.ORAL COMMUNICATIONSREF O153Identification <strong>of</strong> novel target genes <strong>of</strong> Epstein Barr Virus miRNAsMarjolein HOOYKAAS 1 , Lione WILLEMS 1 , Sander VAN HOOFF 2 ,Maaike RESSING 1 , Marian GROOT KOERKAMP 2 , FrankHOLSTEGE 2 , Robert Jan LEBBINK 1 , Emmanuel WIERTZ 11 Department <strong>of</strong> Medical Microbiology, UMC Utrecht, Utrecht, THENETHERLANDS; 2 Microarray Facility, Department <strong>of</strong> Molecular CancerResearch, UMC Utrecht, Utrecht, THE NETHERLANDSMicroRNAs (miRNAs) are posttranscriptional gene regulators that playimportant roles in many cellular processes. These small non coding RNAmolecules bind to complementary target mRNAs, thereby inducing RNAdestabilization and inhibition <strong>of</strong> translation. Several DNA viruses hijackthe cellular miRNA machinery to produce their own miRNAs, which <strong>of</strong>fersopportunities to regulate both cellular and viral gene expression. However,the targets and functions <strong>of</strong> viral miRNAs remain largely unknown. Members<strong>of</strong> the herpesvirus family encode the highest numbers <strong>of</strong> miRNAs.Epstein Barr Virus (EBV), for example, expresses only 1 9 viral proteinsduring latency, but this repertoire is complemented by more than 40 differentmiRNAs. Their lack <strong>of</strong> immunogenicity <strong>of</strong>fers a specific advantageover protein coding genes. EBV miRNAs are abundantly expressed inEBV associated tumors, including nasopharyngeal carcinoma, indicatingthat they may play a role in tumorigenesis.Using a newly developed lentivirus based miRNA expression system, EBVmiRNAs were expressed in the nasopharyngeal cell line HK1. Transcriptomepr<strong>of</strong>iling revealed numerous genes differentially expressed betweenmiRNA positive and negative cells, including previously identified bonafide EBV miRNA targets such as IPO7 and DAZAP2. Amongst the potentialmRNA targets are regulators <strong>of</strong> cell growth and apoptosis and genesthat may impact adaptive and innate immunity. We validated a set <strong>of</strong> thenewly identified genes by using miRNA reporter constructs and confirmedthat they are direct targets <strong>of</strong> specific EBV miRNAs. The role <strong>of</strong> these newmiRNA target interactions in EBV biology is being established, a.o. usinga series <strong>of</strong> newly developed miRNA inhibitors. These inhibitors effectivelycounteract EBV miRNA function and <strong>of</strong>fer new perspectives for thetreatment <strong>of</strong> EBV associated malignancies.REF O154Alternative splicing signatures discriminate ATL cells from untransformedCD4+ counterparts deriving from HTLV 1 infectedindividualsMorgan THENOZ 1 , Céline VERNIN 1 , Christiane PINATEL 2 , NicolasNAZARET 3 , Joel LACHUER 3 , Antoine GESSAIN 4 , DidierAUBOEUF 5 , Eric WATTEL 1 , Franck MORTREUX 11 Oncovi<strong>rologie</strong> et Biotherapies, UMR5239 CNRS/ENS Lyon/UCBL/HCL,Hopital Pierre Benite, Lyon, FRANCE; 2 Oncovi<strong>rologie</strong> et Biothérapies,Centre Léon Bérard, Lyon, FRANCE; 3 Pr<strong>of</strong>ileXpert, Neurobiotec Service,Bron, FRANCE; 4 Unit <strong>of</strong> Epidemiology and Physiopathology <strong>of</strong> OncogenicViruses, Department <strong>of</strong> <strong>Virology</strong>, Institut Pasteur, Paris, FRANCE;5 Institut National de Santé et de Recherche Médicale U590, Centre LéonBérard, Lyon, FRANCEThe clonal expansion and malignant transformation <strong>of</strong> HTLV 1 infectedCD4+ T cells has been linked to the reprogramming effects <strong>of</strong> HTLV 1on host transcriptional pr<strong>of</strong>ile. Coupled to transcription, alternative splicing(AS) is a post transcriptional process that plays critical role in thecomplexity <strong>of</strong> transcriptome and splicing abnormalities frequently occurin cancer. To examine whether AS modifications associate with HTLV1 associated leukemogenesis, we compared the exon expression pr<strong>of</strong>iles<strong>of</strong> ATL cells with that <strong>of</strong> CD4+ T cell clones obtained by limited dilutioncloning <strong>of</strong> PBMC deriving from HTLV 1 carriers. 3 ATL cells and12 untransformed infected clones clustering in infected, uninfected, PHAstimulated or unstimulated CD4+ T cells were compared for exon RNAcontent using Exon Chip Human microarray. Hierarchical clustering analysis<strong>of</strong> data identified 12516 alternative spliced events (3642 genes) thatclearly separated ATL samples from the 4 untransformed phenotypes mentionedabove. Microarray data were confirmed for 18 AS events using exonspecific RT PCR analysis. Pathway analysis <strong>of</strong> alternatively spliced genesin ATL cells revealed new AS based pathways for p53 signaling, cell cycleand DNA replication while those <strong>of</strong> untransformed infected CD4+ T cellswere enriched in pathways for cellular movement and DNA repair. Thesefindings unveil a new layer <strong>of</strong> complexity in the interplay between HTLV 1and host cell gene expression machinery in which AS might play a centralrole in tumor initiation and promotion.Vi<strong>rologie</strong>, Vol 17, supplément 2, septembre 2013S109
5 th <strong>European</strong> <strong>Congress</strong> <strong>of</strong> <strong>Virology</strong>REF O155High risk human papillomavirus E6 induces expression <strong>of</strong> the histonedemethylase KDM2B by repressing miR 146a in human keratinocytesElektra PETA 1 , Alessandro SINIGAGLIA 1,2 , Valentina MILITELLO 1 ,Angela GRASSI 3 , Barbara DI CAMILLO 3 , Marta TREVISAN 1 ,Giorgio PALÙ 1 , Luisa BARZON 11 Department <strong>of</strong> Molecular Medicine, University <strong>of</strong> Padova, Padova,ITALY; 2 IOV Istituto Oncologico Veneto, Padova, ITALY; 3 Department<strong>of</strong> Information Engineering, University <strong>of</strong> Padova, Padova, ITALYHigh risk HPV infection modulates expression <strong>of</strong> cellular microRNAs(miRNAs) involved in cell proliferation and tumorigenesis. MiRNAmicroarray analyses were used to identify alterations in miRNA expressionpr<strong>of</strong>ile in primary cultures <strong>of</strong> human foreskin keratinocytes inducedby expression <strong>of</strong> E6 and E7 <strong>of</strong> HPV16 or HPV6. While HPV6 E6 andE7 expression did not significantly change cellular miRNA expressionpr<strong>of</strong>ile, E6 and E7 <strong>of</strong> HPV16 significantly altered expression <strong>of</strong> some cellularmiRNAs, <strong>of</strong>ten with opposing effects, such as in the case <strong>of</strong> miR 34miRNAs and miR 146a, that were downregulated by E6 and upregulatedby E7. Among the significantly modulated miRNAs, miR 146a, that hasbeen shown to inhibit innate immune response and interferon pathway,was selected for further studies. Overexpression <strong>of</strong> miR 146a in humankeratinocytes and cervical carcinoma cell lines led to decreased cell proliferationand invasiveness. In promoter/luciferase reporter assays in humankeratinocytes, inactivation <strong>of</strong> NF kB trascription factor binding site in themiR 146a promoter abolished luciferase reporter activity independentlyfrom HPV16 E6 and E7 expression, while inactivation <strong>of</strong> IRF3/IRF7 andc Myc binding sites inhibited and induced, respectively, the miR 146a promoterin the presence <strong>of</strong> HPV16 E6. Repression <strong>of</strong> miR 146a promoter by cMyc was confirmed in HPV positive cancer cell lines. KDM2B/FBXL10,a H3K36 specific histone demethylase that is highly expressed in embryonicstem cells and induces pluripotency, was predicted to be novel directtarget for miR 146a and validated experimentally.invasive capacity <strong>of</strong> LMP1 expressing cells through extracellular matrix.Taken together, our data suggest that LMP1 mediated upregulation <strong>of</strong>Fascin contributes to EBV associated pathogenesis.REF O157Cell cycle modulation by Marek’s Disease Virus: the tegument proteinVP22 triggers S Phase arrest and DNA damage in proliferating cellsLaëtitia TRAPP FRAGNET 1 , Danièle VAUTHEROT 1 ,YvesLEVERN 1 , Sylvie REMY 1 , Elisa BOUTET ROBINET 2 , Gladys MIREY 2 ,Jean François VAUTHEROT 1 , Caroline DENESVRE 11 INRA, UMR1282, NOUZILLY, France; 2 INRA/INP, Toxalim, UMR1331,TOULOUSE, FRANCEMany viruses modulate cell cycle progression to enhance their replicationand persistence in the host cell. In the case <strong>of</strong> oncogenic viruses, this processmay ultimately lead to cellular transformation and oncogenesis. Inthe present study, we demonstrate that Marek’s disease virus (MDV), analphaherpesvirus responsible <strong>of</strong> T lymphoma in chickens, is also able tosubvert the cell cycle progression. Infection <strong>of</strong> primary chicken embryoskin cells with MDV triggered the re entry <strong>of</strong> quiescent cells into the cellcycle and delayed the progression <strong>of</strong> the cell cycle into S phase. We couldalso identified the tegument protein VP22 as a major MDV encoded cellcycle regulator since its over expression in proliferating cells led to a dramaticcell cycle arrest in S phase. This striking functional feature <strong>of</strong> VP22appears to be conserved among members <strong>of</strong> the Alphaherpesvirinae andis depended on its nuclear localization and histones binding capacity. Themechanism underlying the VP22 mediated S phase arrest might rely on theability <strong>of</strong> VP22 to generate DNA double strand breaks. Taken together,our results shed a new light on the mechanisms <strong>of</strong> MDV induced lymphomagenesisby describing a new role for the VP22 tegument protein inviral reprogramming <strong>of</strong> the cell cycle <strong>of</strong> the host cell associated to DNAdamage induction.REF O156Induction <strong>of</strong> the tumor marker Fascin by latent membrane protein 1(LMP1) depends on NF KB and could contribute to invasionAndrea K. KRESS 1 , Martina KALMER 1 , Caroline F. MOHR 1 ,Christine GROSS 1 , Melanie C. MANN 1 , Arnd KIESER 2 ,Bernhard FLECKENSTEIN 11 Institute <strong>of</strong> Clinical and Molecular <strong>Virology</strong>, Friedrich Alexander UniversitätErlangen Nürnberg, Erlangen, GERMANY; 2 Research Unit GeneVectors, Helmholtz Zentrum München German Research Center for EnvironmentalHealth, Munich, GERMANYThe actin bundling protein Fascin (FSCN1) is a tumor marker that is highlyexpressed in numerous types <strong>of</strong> cancer including lymphomas and is importantfor migration and metastasis <strong>of</strong> tumor cells. Fascin has also beendetected in B lymphocytes that are freshly infected with Epstein Barrvirus (EBV), however, both the inducers and the mechanisms <strong>of</strong> Fascinupregulation are still unclear. Here we show that the EBV encoded oncoproteinlatent membrane protein 1 (LMP1), a potent regulator <strong>of</strong> cellularsignaling and transformation, is sufficient to induce both Fascin mRNAand protein in lymphocytes. Fascin colocalizes with actin in the cytoplasm<strong>of</strong> LMP 1 transfected Jurkat cells and in lymphoblastoid cell lines. Fascinexpression is mainly regulated by LMP1 via the C terminal activationregion 2 (CTAR2). Block <strong>of</strong> canonical NF KB signaling using a chemicalinhibitor <strong>of</strong> IKB kinase (IKK ) or cotransfection <strong>of</strong> a dominant negativeinhibitor <strong>of</strong> IKBa (NFKBIA) reduces LMP1 induced Fascin expression.Beyond that, chemical inhibition <strong>of</strong> IKK reduces Fascin mRNA levels inEBV transformed lymphoblastoid cell lines, indicating that canonical NFKB signaling is required for LMP1 mediated regulation <strong>of</strong> Fascin both intransfected and transformed lymphocytes. Use <strong>of</strong> specific shRNAs leadsto knockdown <strong>of</strong> LMP1 induced Fascin expression and could influence theREF O158The human bocavirus is associated with lung and colorectal cancersand persists in solid tumoursOliver SCHILDGEN, Verena SCHILDGEN, Monika MALECKI,Ramona Liza TILLMANN, Michael BROCKMANNKliniken der Stadt Köln, Cologne, GERMANYThe human bocavirus (HBoV) is the second human pathogenic parvovirus.It causes respiratory infections and gastroenteritis. Some autonomousanimal parvoviruses and also some human non autonomous parvovirusesare known to persist and even integrate into the host genome resulting intransformation <strong>of</strong> the infected cells and eventually contribute to the multistep development <strong>of</strong> cancer. Also HBoV persists in a so far unknown percentage<strong>of</strong> patients without causing clinical symptoms beyond those <strong>of</strong> theprimary infection. The aim <strong>of</strong> the present study was to analyze the role <strong>of</strong>HBoV in lung and colorectal cancers. Therefore, formalin fixed paraffinembedded archived tumor samples were screened for HBoV DNA by PCR,Southern blotting, and sequencing. Positive tissues were further subjectedto FISH analyses specifically detecting HBoV DNA in the infected cells.In total, 11 <strong>of</strong> 60 (18.3%) lung and 9 <strong>of</strong> 44 (20.5%) colo rectal tumorswere tested positive for HBoV DNA, confirmed by sequencing and FISHanalyses. HBoV DNA thereby is present in the nuclei <strong>of</strong> infected cells,either in single or multiple copies, and appears also to form filaments.Moreover the FISH patterns give rise to the hypothesis that HBoV DNApersists either as cccDNA or integrates into the host genome. This supportsthe further hypothesis that the virus plays an active role in cancer byinteractions with the host genome, or contributes to cancer developmentindirectly by inducing a persisting inflammation, as other DNA viruses likethe human hepatitis B virus do. The occurrence <strong>of</strong> HBoV DNA filamentscould confirm the postulated? or rolling hairpin replication mechanism.S110 Vi<strong>rologie</strong>, Vol 17, supplément 2, septembre 2013
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