rologie i - European Congress of Virology

5 th European Congress of Virologypromoters, to repress its own promoter, and to modulate the host cellcycle. Since the molecular aspects of its functioning still remain to befully elucidated, taking advantage of a specific IE2 inhibitor termedWC5, we investigated deeper the mechanisms of IE2 regulatory activityon responsive promoters. By mutagenesis, cell based assays, and electrophoreticmobility shift assays, we demonstrated that the abortive effectexerted by the compound on HCMV replication is due to the interferencewith the interaction between IE2 and different DNA sequences locatedwithin responsive viral promoters. Moreover, by focusing on a prototypicEarly promoter, i.e., the DNA polymerase gene promoter, we analysedthe contribution of different DNA elements in its transactivation andidentified two previously uncharacterised sequences bound by IE2 invitro, involved in UL54 promoter transactivation in transfected cells,and that may serve for the recruitment of IE2 at the promoter. Our studycontributes in shedding light on the molecular aspects of IE2 activity asan essential virus encoded transcription factor.REF 189DNA methylation: A key regulator of Marek’s disease virus life cycle?Carole MIGNION 1 , Isabelle GENNART 1 , Damien COUPEAU 1 , SylvieLAURENT 2 , Ginette DAMBRINNE 2 , Denis RASSCHAERT 2 , BenoîtMUYLKENS 11 Integrated Veterinary Research Unit (IVRU), Namur, BELGIUM;2 Transcription Lymphome Viro Induit (TLVI), Tours, FRANCEMarek’s disease is a lymphoproliferative disease, induced by an avianherpesvirus, Marek’s disease virus (MDV). MDV presents two alternativestages of infection: lytic and latent. During latency, only few transcripts,involved in the maintenance of the latency phase and/or in tumerogenesis,are expressed. During the lytic phases, latency transcripts are repressedwhile others genes involved in the lytic phase are reactivated. In this study,we investigated the role of DNA methylation in the regulation of the geneexpression switch observed in the alternative stages of MDV infection.Five genes related with lytic (pp38, ICP4 and ICP27) and latent (clustermicroRNA 9/4 and vTR) phases were selected. Firstly, the impact of DNAdemethylation was assessed on pp38, ICP27 and miR9/4 transcript levelsin latently infected cells. The viral genome demethylation induced a sharpincrease in the expression level of the three genes, showing strong associationbetween DNA methylation patterns and promoter activities. Then,the promoters were analyzed to determine their DNA methylation patternthrough Bisulfite Genomic Sequencing Analysis at relevant in vitro andin vivo conditions of MDV infection. For pp38, ICP4 and vTR promoters,the CpG methylation level is high (80%) in latency while it is lower (18%)during reactivation and presents 0% in lytic infection. On the contrary,miR4 5p promoter shows 0% in the three conditions. In vivo studies onperipheral blood leucocyte infected with MDV revealed that ICP4, ICP27and vTR promoters display a DNA methylation percentage close to 0%.REF 190Investigation of influenza A virus M segment splicing controlOlivier MONCORGÉ 1 , Anita ARTARINI 2 , Alexander KARLAS 2 ,Thomas MEYER 2 , Chad M SWANSON 3 , Michael H MALIM 3 , WendyS BARCLAY 11 Section of Virology, Department of Medicine, Imperial College London,London, UNITED KINGDOM; 2 Molecular Biology Department, MaxPlanck Institute for Infection Biology, Berlin, GERMANY; 3 Department ofInfectious Diseases, Kings College London School of Medicine, London,UNITED KINGDOMInfluenza virus, as an obligatory intracellular parasite, relies on host cellfunctions for various aspects of its replication cycle. Splicing is one ofthe key cellular functions essential for influenza virus replication. Humangenes typically include introns, which are removed by the splicing machinery.Two of the viral gene segments (M and NS) produce spliced andunspliced viral mRNAs. Regulation of M and NS genes splicing is currentlynot well understood. It is believed that both virus and cellular factorsare involved in the kinetics of influenza virus mRNA splicing, and inefficientsplicing control has been suggested to be responsible for abortivereplication in some models. We developed a cell based influenza polymerasedriven minireplicon assay that monitors expression of three mRNAsproduced from the M gene: M1 (unspliced) and M2 and m3 (spliced).Using this assay, we tested whether the nature of the viral polymerasethat directs viral mRNA transcription affects the efficiency of splicing andalso confirmed that the viral protein NS1 modulates M gene pre mRNAsplicing. Investigating a role for the host cell in influenza splicing control,we compared M gene splicing in cell types of mammalian and avian originsand investigated the role of some cellular factors on M gene splicingcontrol. We also tested in this system some known small molecule inhibitorsof the splicing machinery and concluded that this assay can also beused to screen for novel inhibitors of splicing that might control influenzareplication and from which the virus could not escape.REF 191The metabolic sensors FXRa, PGC 1a and SIRT1 cooperativelyregulate hepatitis B virus transcriptionChristophe RAMIÈRE 1,2,3 , Claire CURTIL 2,3 , Liviu SorinENACHE 2,3,4 , Pauline RADREAU 2,3 , Anne Gaëlle DRON 1,2,3 , CarolineSCHOLTÈS 1,2,3 , Alexandre DELOIRE 2,3 , Didier ROCHE 5 , VincentLOTTEAU 2,3 , Patrice ANDRÉ 1,2,31 Hospices Civils de Lyon, Lyon, FRANCE; 2 INSERM U1111, CIRI, Lyon,FRANCE; 3 Université de Lyon, Lyon, France; 4 University of Medicineand Pharmacy, TârguMure, ROMANIA; 5 Edelriss.a.s., Lyon, FRANCEHepatitis B virus (HBV) transcription is highly dependent on liver enrichednuclear receptors involved in metabolic regulation. Among thesenuclear receptors, the bile acid receptor farnesoid X receptor a (FXRa) hasbeen shown to enhance HBV core promoter activity and pregenomic RNAsynthesis. Interestingly, two FXRa modulators, peroxisome proliferatoractivated receptor coactivator 1 a (PGC 1a) and the deacetylase SIRT1have been detected associated to HBV genomes ex vivo. Moreover, PGC1a induction during fasting has been shown to increase HBV replication inmice liver. SIRT1 is also activated in the fasted state. However its impact onHBV life cycle is currently unknown. In this study, we investigated the roleof SIRT1 activation on the regulation of HBV transcription and replicationin vitro. Using a luciferase reporter system, we first demonstrated thatSIRT1 activation increases the transcription from the core promoter regionthrough an FXRa and PGC 1a dependent mechanism. Moreover, we showedin cells transfected with a full length HBV genome, that FXRa, PGC1a and SIRT1 cooperate to enhance pregenomic RNA synthesis and covalentlyclosed circular DNA accumulation. We thus identified a subnetwork,composed of the nuclear receptor FXRa, the transcriptional coactivatorPGC 1a and the energy sensor SIRT1, which increases HBV replication.Each of these proteins is a key regulator of physiological metabolism,strengthening the hypothesis that HBV replication is tightly controlled byenvironmental stimuli, very similarly to metabolic genes.REF 192In vivo transcriptomic study of 39 viral genes and 17 host genes inPacific oyster, Crassostrea gigas infected by ostreid herpesvirus 1:comparing two oyster families presenting different levels of susceptibilityAmelie SEGARRA, Nicole FAURY, Florian MAUDUIT, PhilippeHAFFNER, Jean François PEPIN, Delphine TOURBIEZ, SuzanneTRANCARD, Agnes TRAVERS, Pierrick MOREAU, TristanRENAULTIfremer LGPMM, La Tremblade, FRANCES172 Virologie, Vol 17, supplément 2, septembre 2013