rologie i - European Congress of Virology

5 th European Congress of VirologySince 1991, high rates of mortality among Pacific oysters spat have beenassociated with the detection of a herpesvirus called ostreid herpesvirus1, (OsHV 1) and reported in different countries, including France. However,no study has been performed to understand and follow viral geneexpression during the viral infection whereas gene expression in otherherpesviruses was widely studied. An in vivo transcriptomic study wasperformed during an OsHV 1 infection in Crassostrea gigas spat in orderto better undrstand interactions between the host and its pathogen. In thiscontext, 39 viral genes and 17 host genes were selected and analysed byreal time PCR using two oyster families presenting different levels of susceptibiltyto the virus. First virus RNAm transcripts were detected at 8 hpost injection (hpi) in the most susceptible family whereas in the less susceptibleone first transcripts were observed at 12 hpi. After 12 hpi 39 viralgenes were detected in the most susceptible family. However, in the lesssusceptible family, RNA transcripts of all the 39 virus genes were detectedonly at 26 hpi. In addition, analysis of the relative expression of the hostgene encoding the Myeloid differentiation factor 88 showed an up expressionin the most susceptible family at 26 h pi. This study focused on thedetecion of RNAm transcripts in Crassostrea gigas spat challenged withOsHV 1. First results confirm the replication of the virus during infectionand therefore information on the viral life cycle of OsHV 1 and the defensemechanisms against the virus can be drawn.REF 193The effect of coxsackievirus A9 (CAV9) infection on the nucleusproteins PSF, SC35 and PML, located in the nuclear paraspeckles,speckles and PML bodies respectivelyAshjan SHAMI, Glyn STANWAY, Maysoon MUTABAGANIUniversity of Essex, Colchester, UNITED KINGDOMPicornaviuses replicate in the cytoplasm, but there is growing evidencethat the cell nucleus is affected by infection e.g. transcription factor cleavage,relocation of nuclear proteins and alteration to nucleo cytoplasmicshuttling. The nucleus has a number of sub domains and structures, someof which have been shown to be affected by virus infection. We have previouslyobserved that human parechoviruses affect the distributions of thenuclear paraspeckle protein PSF, but not proteins associated with PMLbodies or nuclear speckles. We are currently studying whether enteroviruseshave the same effect, using coxsackie virus A9 (CAV9), to determinewhether this effect on nuclear paraspeckles and lack of an effect on specklesand PML bodies are general features of picornavirus infections. Thisis being studied using EGFP PSF, EGFP SC35 and EGFP PML fusionsand antibodies to these nuclear proteins to investigate the timescale of relocalisation,how the virus achieves any change and the role these changesplay in enteroviruses and parechvirus replication.REF 194Characterization of the translational mechanism that controls thesynthesis of the short form of HCV core+1/ARFP proteinNiki VASSILAKI 1 , Ioly KOTTA LOIZOU 1 , PanagiotisSAKELLARIOU 1 , Ralf BARTENSCHLAGER 2 , PenelopeMAVROMARA 11 Hellenic Pasteur Institute, Molecular Virology, Athens, GREECE;2 University of Heidelberg, Department of Infectious Diseases, MolecularVirology, Heidelberg, GERMANYPrevious studies from our laboratory and others have shown that HepatitisC Virus (HCV) genome, in addition to the polyprotein precursor openreading frame (ORF), possesses a second functional ORF overlapping thecore gene that encodes a protein initiated by internal translation initiationat codons 85/87, known as core+1/ARFP/S (short). To investigate themolecular mechanism that controls core+1/S synthesis, we searched fora putative IRES like element within the core coding sequence. For this,different parts of the 1a/2a core coding sequences were tested for theirputative IRES activity after insertion in a dual luciferase reporter construct.In vitro transcribed RNAs derived from these cassettes were transfectedinto Huh7 cells. The results showed the presence of a functional regulatoryelement within the 5 ′ end of core/core+1 coding sequence that coulddirect internal translation initiation of the core+1/ARFP. This element iscomprised of the core nucleotides located upstream of the core+1 initiatorcodons 85/87. The integrity of the conserved core RNA structures stemloop SL47 and SL87 is essential for this IRES activity. Interestingly, inthe JFH1 replicon system, core+1/S is the predominant isoform of core+1protein at early hours of viral translation/replication cycle and is expressedindependently of a functional viral IRES or polyprotein initiator whereashighly correlated with intact stem loops SL47 and SL87. At late hours ofvirus replication cycle, core+1/short is coexpressed with a longer core+1isoform that is translated in a IRES dependent manner.REF 195Investigating the interactions between the FMDV Lbpro and hostfactor eIF4GIIMartina AUMAYR 1 , Sofiya FEDOSYUK 1 , Georg KONTAXIS 2 ,TimSKERN 11 Max F. Perutz Laboratories, Medical University of Vienna, Vienna,AUSTRIA; 2 Max F. Perutz Laboratories, University of Vienna, Vienna,AUSTRIAThe picornaviral foot and mouth disease virus (FMDV) is the causativeagent of a highly contagious disease affecting cloven hoofed animals.Upon infection, the single stranded RNA genome is released into thecytoplasm and interacts with the host cell translation machinery. In orderto achieve efficient replication of their own proteins, picornavirusesinterfere with cellular processes. Thus, FMDV hijacks the host celltranslation machinery by cleaving the eukaryotic initiation factor (eIF)4Gleading to the shut off of host cell protein production. eIF4G cleavage isperformed by the leader protease (Lbpro). Picornaviral cleavage of 4G isa determinant of virulence, but has not yet been elucidated structurally.We are therefore investigating how the Lbpro interacts with 4G. Weshowed that 4E enhances the cleavage of 4G by the Lbpro in an in vitroassay. A complex of 4G and Lbpro is not detectable on size exclusionchromatography. However, when 4E is present a complex of all threeproteins is stably formed. Experiments with surface plasmon resonanceshowed similar results and confirmed that the dissociation constant of the4G 4E complex with the Lbpro is about 10 fold lower than that for eachsingle protein to the Lbpro. The single mutation C133S near the LbproC terminus influences the 4G cleavage by reducing Lbpro binding tothe 4G/4E complex. We have started to elucidate the structure of the 4Gfragment by means of two and three dimensional NMR experiments.REF 196Interaction of tick borne encephalitis virus and host cell on proteinsynthesis levelHana TYKALOVÁ 1,2 , Zuzana VAVRUŠKOVÁ 2 , Jirí CERNÝ 1,2 , LiborGRUBHOFFER 1,21 Faculty of Science, University of South Bohemia, Ceské Budejovice,CZECH REPUBLIC; 2 Biology Centre, Institute of Parasitology, CzechAcademy of Sciences, Ceské Budejovice, CZECH REPUBLICTick borne encephalitis virus (TBEV) is a lurking menace that endangerslives of thousands of people annually. In order to accomplish their lifecycle successfully, viruses must compete with host cell at many levelsand need to hijack host cell machinery. To determine an unknown aspectof TBEV pathogenesis we focused on the virus interaction with host cellprotein synthesis pathway in human neural cells. Several aspects wereevaluated. We studied the onset of TBEV protein synthesis in the host cellsVirologie, Vol 17, supplément 2, septembre 2013S173