rologie i - European Congress of Virology

5 th European Congress of VirologyPOSTERS01. ONCOGENIC MECHANISMS OFVIRUSESPosters: REF 001 to REF 008deleted at the expected sites. The EBER 1 deletion mutants have beencloned into EBER expression plasmids carrying the EBV oriP and hygromycin/ampicillinresistance genes. These plasmids have now been stablytransfected into 293T cells and a Burkitt’s lymphoma cell line (BL30) andtheir effect on cell proliferation and apoptosis will be presented.Understanding the function of EBERs may shed light on the molecularmechanism(s) by which EBV induces tumorigenesis.REF 001Selective activation of non canonical NF kappaB through a uniqueTRAF 3 binding motif in the viral oncoprotein TioBrigitte BIESINGER 1 , Sarah Jill DE JONG 1,4 , Jens ChristianALBRECHT 1 , Fabian GIEHLER 2 , Arnd KIESER 2 , Heinrich STICHT 31 Virologisches Institut, Friedrich Alexander Universitaet, Erlangen,GERMANY; 2 Abteilung Genvektoren, Helmholtz Zentrum Muenchen,Muenchen, GERMANY; 3 Institut fuer Biochemie, Friedrich AlexanderUniversiaet, Erlangen, GERMANY; 4 St. Giles Laboratory of Human Geneticsof Infectious Diseases, The Rockefeller University, New York, USAHerpesvirus ateles is capable to transform human T cells to long termgrowth in culture. Its oncoprotein Tio (Two in one) transfers this phenotypeto a transformation deficient recombinant herpesvirus saimiri. Oligomeric,membrane anchored Tio constitutes a signaling platform that recruitsSrc family kinases to activate STAT factors and TRAF6 to activate canonicalNF kappaB. Independent of these interaction partners, Tio inducesNIK stabilization and p100 processing, the hallmarks of non canonicalNF kappaB activation, through an as yet unidentified mechanism. We nowidentified a distinct sequence motif in Tio that specifically recruits TRAF3,a negative regulator of non canonical NF kappaB signaling. Through thisunique motif, Tio triggers a ubiquitin independent depletion of TRAF3from the cytosol and thereby releases the intrinsic activity of the non canonicalNF kappaB signaling cascade leading to p100 processing. Notably,mutation of the TRAF3 binding site does not affect canonical signalingintermediates and target gene regulation. Thus, Tio targets TRAF3 toinduce non canonical NF kappaB independent of cross talk into the canonicalcascade. In summary, we have identified a new and unique type ofTRAF3 binding motif that selectively mediates non canonical NF kappaBactivation and discloses studies into the specific functions of this pathwayand its role in T cell growth regulation.REF 002Creation of Epstein Barr virus encoded small RNA (EBER) mutantsand analysis of their functionGulfaraz KHAN, Pretty PHILIP, Waqar AHMEDUAE University, College of Medicine, Al Ain, UAEEpstein Barr virus (EBV), an oncogenic lymphotropic herpesvirus is implicatedin the pathogenesis of a number of human malignancies. Althoughthe mechanism by which EBV leads to cell transformation is not clear, anumber of viral latent gene products, including non protein coding smallRNAs have been implicated in the transformation/proliferation process.EBER 1 and EBER 2 are two such RNA molecules which are abundantlyexpressed in all EBV infected cells, but their function is poorly understood.Both of these RNA molecules have a secondary structure and are boundto cellular proteins. We set out to investigate the functions of EBER 1 bycreating EBER 1 mutants. We used spliced overlap extension PCR (SOEPCR) approach to create EBER 1 mutants with deletions in regions normallybound to cellular proteins. Using this approach, we have successfullydeleted stem loops (SL) 1, 3 and 4 of EBER 1. The subsequent mutantswere cloned and sequenced to verify that the intended target regions wereREF 003Human cytomegalovirus activates pro oncogenic pathways in primaryhuman hepatocytes and HepG2 cellsAmit KUMAR, Wasim ABBAS, Quentin LEPILLER, GeorgesHERBEINUniversity of Franche Comté, Besançon, FRANCEBackground: In the recent past, there has been increased concern in thepotential involvement of human cytomegalovirus (HCMV) in carcinogenesis.HCMV seroprevalence was enhanced in patients with hepatocellularcarcinoma (HCC) however, a possible link between HCC and HCMVinfection remains to be assessed. The aim of this work was to investigatethe protumor influence of HCMV on primary human hepatocytes (PHH)and HepG2 cells. Methods: Following infection of PHH and HepG2 cellsby two different strains of HCMV, we measured the production of IL 6 inculture supernatants by ELISA and the protein levels of STAT3, pSTAT3,JAK, cyclin D1, survivin, p53, p21, and Mdm2 by western blotting ininfected and uninfected cells. Ki67Ag expression was measured as a proliferationmarker whereas transformation was probed using soft agar colonyformation and tumorsphere assay. Results: PHH and HepG2 cells infectedwith HCMV resulted in the production of IL 6 and the subsequent activationof the IL 6R JAK STAT3 pathway. Expression of Cyclin D1, Survivinand Ki67Ag was upregulated by HCMV despite a paradoxical overexpressionof p53 and p21. Notably, we observed the formation of coloniesin soft agar seeded with PHH infected with HCMV and 2.5 fold moretumorspheres in HCMV infected HepG2 cultures than in uninfected ones.Conclusion: HCMV activated the IL 6 JAK STAT3 pathway in PHH andHepG2 cells, favored cellular proliferation, induced PHH transformationand enhanced HepG2 tumorsphere formation. Our observations raise thepossibility that HCMV infection might be involved in the onset of HCC.REF 004MicroRNA profiling in E6/E7 HPV transformed human keratinocytesGiorgio MANGINO 1 , Maria Vincenza CHIANTORE 2 , MarcoIULIANO 1 , Maria Simona ZANGRILLO 1 , Gabriele VACCARI 3 , RositaACCARDI 4 , Massimo TOMMASINO 4 , Gianna FIORUCCI 2,5 ,Giovanna ROMEO 1,51 Sapienza University of Rome/Dept. of Medico Surgical Sciences andBiotechnologies, Latina, ITALY; 2 Istituto Superiore di Sanità/Dept. ofInfectious, Parasitic and Immune mediated Diseases, Rome, ITALY;3 Istituto Superiore di Sanità/Dept. of Veterinary Public Health andFood Safety, Rome, ITALY; 4 International Agency for Research onCancer WHO/Infections and Cancer Biology Group, Lyon, FRANCE;5 CNR/Institute of Molecular Biology and Pathology, Rome, ITALYMicroRNAs (miRNAs) are naturally occurring noncoding RNAs that playa key role in gene regulation. They are highly conserved single strandedRNAs (∼22 nucleotides) that are cleaved from larger precursor transcripts.miRNAs exert their regulatory effects by cleaving the mRNAtargets or repressing their translation and are predicted to regulate at leastone third of all human genes. This posttranscriptional gene regulationhas been demonstrated to be active in fundamental cellular processes ascell proliferation, differentiation, and death. miRNAs are also involvedin the development and progression of human diseases, such as cancer.S118 Virologie, Vol 17, supplément 2, septembre 2013