rologie i - European Congress of Virology

5 th European Congress of Virologyanalysis and deep sequencing to investigate KSHV miRNA expression.Using two cellular contexts, a B cell line infected by KSHV and a stable cellline with a genomic integration of the KSHV miRNA cluster, we identifieddifferential accumulation of the KSHV miRNAs. Our results suggest thatthe expression of the viral miRNAs is post transcriptionally regulated. Wefocus on factors influencing the regulation, like structural features of thepri miRNA or accessory proteins important for the expression of miRNAs.We determined the secondary structure of KSHV pri miRNAs by SHAPE(Selective 2’ Hydroxyl Acylation analyzed by Primer Extension). Then,we verified if the pre miRNA stem loops were optimal Drosha substratesand we tried to identify regulatory elements required for the expression ofKSHV miRNAs.chemiluminescent (CL) detection have been developed to quantify viralDNA, NS and VP1 2 transcripts, and NS and VP1 2 proteins followingUT7/EpoS1 infection. CL microscope imaging methods allowed evaluatingthe fraction of infected cells and the amount of viral nucleic acidswithin each single cell. In our experimental conditions, B19 transcriptionalactivity, leading to the production of both NS and VP mRNAs, hasbeen detected early in viral cycle, in increasing fractions of cells from 12hpi, and before genome replication, evident from 24 hpi. NS protein hasbeen detected mainly at early stages of infection, while the capsid proteinsaccumulate within cells only at 48 72 hpi, suggesting an inhibitory effecton capsid protein translation as a post transcriptional regulation event.REF 185Characterization of T Antigens encoded by Trichodysplasia spinulosaassociated polyomavirus; Evidence for Middle T ExpressionMariet FELTKAMP, Christina DARGEL, Els VAN DER MEIJDENLeiden University Medical Center, Leiden, THE NETHERLANDSIn 2010 we identified the Trichodysplasia spinulosa associated polyomavirus(TSPyV) in a patient with Trichodysplasia spinulosa (TS), a follicularskin disease of immunocompromized patients. Like other polyomaviruses(PyVs), the genome of TSPyV can be divided in an early (T) and late (VP)coding region. T antigens play key roles in viral DNA replication andtranscription, and in some cases are responsible for cellular transformation.The T antigen mRNAs are alternatively spliced to encode at least twoproteins, Small T (ST) and Large T (LT), respectively. For some PyVs upto five T transcripts have been identified including one coding for MiddleT (MT), as shown for the murine and hamster PyV. To characterize theT antigens encoded by TSPyV, the complete early region of TSPyV wascloned into pcDNA3 and transiently expressed in HeLa and 293T cells.Sequencing of RT PCR products revealed five different transcripts: T1 (STlike), T2 (LT like), T3 (17kT/57kT like), T4 (MT like) and T5 (ST* like).Transcripts T2, T3 and T4 or T5 (similar expected size) were confirmedby northern blotting. Experiments to confirm protein expression of theidentified T antigen transcripts, in transfected cell lysates and in clinicalsamples, are in progress. In summary, for TSPyV a transcription patternwas found that largely overlaps with other human PyVs. In addition a MTtranscript was identified so far unique for human PyVs. To what extendthe identified T transcripts result in protein expression is the subject ofcurrent study. When obtained, these data will be presented as well.REF 186Analysis on Parvovirus B19 life cycle at single cell level by chemiluminescentimaging assaysGiorgio GALLINELLA 1 , Francesca BONVICINI 1 , Gloria BUA 1 ,Elisabetta MANARESI 1 , Mara MIRASOLI 2 , Martina ZANGHERI 2 ,Aldo RODA 21 Department of Pharmacy and Biotechnology, University of Bologna,Bologna, ITALY; 2 Department of Chemistry, University of Bologna, Bologna,ITALYHuman Parvovirus B19 (B19) is a single stranded DNA virus whosegenome encodes a multifunctional non structural protein (NS), two capsidproteins (VP1, VP2) and additional small non structural proteins (7.5, 9.0and 11 kDa). B19 genome expression has been extensively investigated bymeans of techniques, such as quantitative PCR based assays, that requireextracted nucleic acids from a large number of cells. As a consequence, thedata obtained represent a mean value within cell population. Consideringthat B19 replication process is highly restrictive within the same cell population,methodologies allowing analysis at single cell level could representpotent tools for the investigation of cell virus interactions. In the presentstudy, in situ hybridization and immunocytochemical assays exploitingREF 187Down regulation of HCV Ires dependent translation by nS5a ismediated by PkrEirini KARAMICHALI, Eliza TSITOURA, Peli FOKA, UraniaGEORGOPOULOU, Penelope MAVROMARAHellenic Pasteur Institute, Athens, GREECETranslation initiation of the HCV genome is cap independent and is drivenby an internal ribosome entry site (IRES), located mainly within the5 ′ non coding region. Different data suggested that different viral proteinscan regulate the activity of the HCV IRES as part of the virus’s strategies tocontrol its life cycle. Among them, HCV NS5A protein negatively modulatesthe HCV IRES activity in a specific specific and dose dependentmanner in HepG2 cells. PKR is the RNA activated protein kinase andmediates an anti viral response in humans. PKR plays multiple roles incells, in response to different stress situations. It is previously reportedthat HCV IRES can activate PKR through its binding at domain II ofthe HCV IRES. NS5A protein through the IFN sensitivity determiningregion (ISDR) may mediate IFN resistance and through a direct interactionwith the protein kinase catalytic domain seems to repress PKRactivity. The aim of this study is to investigate whether HCV NS5A downregulation of HCV dependent translation is mediated through PKR. Toaddress this issue, transient transfection was carried using different plasmidvectors either expressing a bicistronic transcriptional unit carrying thechloramphenicol acetyltransferase (CAT) and the firefly luciferase (LUC)genes separated by the HCV or EMCV IRESs or expressing separatelyHCV NS5A and PKR proteins. Induction of PKR was performed by RNAtransfection using polyI;C or interferon. Silencing of PKR was performedusing specific siRNA. We present data concerning the effect of PKR inHCV IRES dependent translation in hepatoma cells as well as, the effect ofNS5A on the modulation of PKR. We provide evidence that HCV NS5Aprotein represses HCV IRES activity through inactivation of the PKR.REF 188The analysis of the inhibition of human cytomegalovirus ImmediateEarly 2 protein by a small molecule reveals interference with DNAbinding and new target sites on a responsive promoterBeatrice MERCORELLI 1 , Anna LUGANINI 2 , Serena MASSARI 3 ,Oriana TABARRINI 3 , Santo LANDOLFO 2 , Giorgio GRIBAUDO 4 ,Giorgio PALÙ 1 , Arianna LOREGIAN 11 Department of Molecular Medicine, University of Padua, Padua, ITALY;2 Department of Public Health and Microbiology, University of Turin,Turin, ITALY; 3 Department of Chemistry and Technology of Drugs, Universityof Perugia, Perugia, ITALY; 4 Department of Life Sciences andSystems Biology, University of Turin, Turin, ITALYHuman cytomegalovirus (HCMV) Immediate Early 2 (IE2) protein is amulti tasking, essential protein which regulates both viral and cellulargene expression. By protein protein interactions and by direct bindingto specific DNA sequences, IE2 is able to transactivate responsiveVirologie, Vol 17, supplément 2, septembre 2013S171