rologie i - European Congress of Virology

5 th European Congress of VirologyREF O155High risk human papillomavirus E6 induces expression of 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 of Molecular Medicine, University of Padova, Padova,ITALY; 2 IOV Istituto Oncologico Veneto, Padova, ITALY; 3 Departmentof Information Engineering, University of Padova, Padova, ITALYHigh risk HPV infection modulates expression of cellular microRNAs(miRNAs) involved in cell proliferation and tumorigenesis. MiRNAmicroarray analyses were used to identify alterations in miRNA expressionprofile in primary cultures of human foreskin keratinocytes inducedby expression of E6 and E7 of HPV16 or HPV6. While HPV6 E6 andE7 expression did not significantly change cellular miRNA expressionprofile, E6 and E7 of HPV16 significantly altered expression of some cellularmiRNAs, often with opposing effects, such as in the case of 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 of miR 146a in humankeratinocytes and cervical carcinoma cell lines led to decreased cell proliferationand invasiveness. In promoter/luciferase reporter assays in humankeratinocytes, inactivation of NF kB trascription factor binding site in themiR 146a promoter abolished luciferase reporter activity independentlyfrom HPV16 E6 and E7 expression, while inactivation of IRF3/IRF7 andc Myc binding sites inhibited and induced, respectively, the miR 146a promoterin the presence of HPV16 E6. Repression of 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 of LMP1 expressing cells through extracellular matrix.Taken together, our data suggest that LMP1 mediated upregulation ofFascin 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 of oncogenic viruses, this processmay ultimately lead to cellular transformation and oncogenesis. Inthe present study, we demonstrate that Marek’s disease virus (MDV), analphaherpesvirus responsible of T lymphoma in chickens, is also able tosubvert the cell cycle progression. Infection of primary chicken embryoskin cells with MDV triggered the re entry of quiescent cells into the cellcycle and delayed the progression of 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 of VP22appears to be conserved among members of the Alphaherpesvirinae andis depended on its nuclear localization and histones binding capacity. Themechanism underlying the VP22 mediated S phase arrest might rely on theability of VP22 to generate DNA double strand breaks. Taken together,our results shed a new light on the mechanisms of MDV induced lymphomagenesisby describing a new role for the VP22 tegument protein inviral reprogramming of the cell cycle of the host cell associated to DNAdamage induction.REF O156Induction of 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 of Clinical and Molecular Virology, 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 of cancer including lymphomas and is importantfor migration and metastasis of tumor cells. Fascin has also beendetected in B lymphocytes that are freshly infected with Epstein Barrvirus (EBV), however, both the inducers and the mechanisms of Fascinupregulation are still unclear. Here we show that the EBV encoded oncoproteinlatent membrane protein 1 (LMP1), a potent regulator of cellularsignaling and transformation, is sufficient to induce both Fascin mRNAand protein in lymphocytes. Fascin colocalizes with actin in the cytoplasmof LMP 1 transfected Jurkat cells and in lymphoblastoid cell lines. Fascinexpression is mainly regulated by LMP1 via the C terminal activationregion 2 (CTAR2). Block of canonical NF KB signaling using a chemicalinhibitor of IKB kinase (IKK ) or cotransfection of a dominant negativeinhibitor of IKBa (NFKBIA) reduces LMP1 induced Fascin expression.Beyond that, chemical inhibition of IKK reduces Fascin mRNA levels inEBV transformed lymphoblastoid cell lines, indicating that canonical NFKB signaling is required for LMP1 mediated regulation of Fascin both intransfected and transformed lymphocytes. Use of specific shRNAs leadsto knockdown of 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 of the infected cells and eventually contribute to the multistep development of cancer. Also HBoV persists in a so far unknown percentageof patients without causing clinical symptoms beyond those of theprimary infection. The aim of the present study was to analyze the role ofHBoV 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 of 60 (18.3%) lung and 9 of 44 (20.5%) colo rectal tumorswere tested positive for HBoV DNA, confirmed by sequencing and FISHanalyses. HBoV DNA thereby is present in the nuclei of 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 of HBoV DNA filamentscould confirm the postulated? or rolling hairpin replication mechanism.S110 Virologie, Vol 17, supplément 2, septembre 2013