rologie i - European Congress of Virology

5 th European Congress of Virologythe ER and late Golgi, respectively. We found that the sequence at the cleavagesite (RRLL of LASV GPC and RRLA of LCMV GPC) is crucial forsubcellular cleavage localization. In order to assess SKI 1/S1P processingin specific subcellular compartments in live cells, we have implementeda novel cell based fluorescence sensor assay (CLIP) that consists of twofluorophores divided by a bait sequence (CLIP RRLL or CLIP RRLA)combined with compartment specific targeting signals. This assay allowsthe visualization of the specific location of SKI 1/S1P activity against adefined bait sequence in live cells. This cell based assay is further suitableto screen for inhibitors of SKI 1/S1P with compartment specific activityin a high throughput format.REF 234Niclosamide and curcumin are potent inhibitors of arenavirus infectionAntonella PASQUATO 1 , Florian ZOPPI 1 , Joel RAMOS DA PALMA 1 ,ANGGAKUSUMA 2 , Eike STEINMANN 2 , Stefan KUNZ 11 Institute of Microbiology, Lausanne University Hospital and Universityof Lausanne (CHUV), Lausanne, SWITZERLAND; 2 Institute of ExperimentalVirology, Twincore Centre for Experimental and Clinical InfectionResearch; a joint venture between t, Hannover, GERMANYViral hemorrhagic fevers by arenaviruses are devastating emerging humandiseases. The lymphocytic choriomeningitis virus (LCMV) is the prototypicmember of the arenavirus family. Currently, no FDA approved vaccineor drugs are available against arenaviruses, with the exception of Ribavirin,with limited efficacy. Small molecules with already known mechanismsof action against pathogens are attractive candidates against arenavirusinfections. Curcumin is a small compound isolated from the spice turmeric.It has a variety of beneficial properties, including antiinflammatoryand chemotherapeutic activities. Curcumin is also known as anti viral viadifferent mechanisms of action. Other small organic compounds extractedfrom plants (e.g. green tea catechins) have antiviral properties. Niclosamideis a FDA approved drug against helminthes with antiviral activity dueto its ability to deplete endosomal proton gradients. Here, we show that curcuminand niclosamide are potent inhibitors of LCMV. Curcumin showsvirucidal activity against LCMV with micromolar range IC50. It blocksLCMV primary infections and reduces virus cell to cell propagation. Similarly,niclosamide potently blocks LCMV infection by interference withthe entry step of the virus life cycle. The mechanism of action relies on theability to negatively tune the pH of late endosomes where LCMV fusiontakes place. Low cost drugs such as curcumin and niclosamide that can bedelivered orally and stored at room temperature in tropical climates mayrepresent promising antiarenavirus candidates.REF 235Post translational modification of CCHF non structural glycoproteinsOlivier REYNARD 1 , Ilona NOVAKOVA 1 , Christophe PEYREFITTE 2 ,Viktor VOLCHKOV 11 INSERM U1111 CIRI, Lyon, FRANCE; 2 IRBA, Lyon, FRANCECrimean Congo Haemorrhagic (CCHF) virus belongs to the bunyavirdae,a family of negative strand segmented RNA viruses responsible of severehaemorrhagic fever in man. Pre or post exposure treatments against thedisease are not yet available for human use. The Hyaloma ticks are thenatural reservoir of the virus and most infections occur after tick’s biteand after spread as a nosocomial disease. The M segment of the genomicRNA encodes a polyprotein precursor that is processed by cellularproteases into several structural and non structural proteins. Role of thenon structural proteins of CCHF as well as their posttranslational processingare not yet clearly defined. In this study we investigate processingof non structural protein precursor GP85 which in the virus infected cellsis believed to give rise upon a cleavage by furin to the so called mucinprotein and GP38. We demonstrate that expression of a GP85 constructin HEK293T cells results in appearance of several forms as detected byanti GP38 antibodies and they are characterized by different mobility onSDS PAGE gels (160kDA, 100kDa and 38kDa). We showed that glycosylationof residues nearby the furin cleavage site are crucial for efficiencyof cleavage and this cleavage is cell type dependent. We demonstrated thatthe 160kDa form cannot be explained neither by a glycosylation patternnor by formation of a conventional dimers since its resistance to reducingconditions. Interestingly, We found that mucin protein was able to conferto any non related fused protein a dimer pattern in reducing conditions.REF 236Ebola virus: transcriptional RNA editing versus premature poly adenylationValentina VOLCHKOVA, Jaroslav VORAC, Viktor VOLCHKOVMolecular Basis of Viral Pathogenicity, CIRI, INSERM U1111, UniversitéLyon 1, Lyon, FRANCEEbola virus (EBOV), a member of the Filovirus family causes lethal hemorrhagicfever in man. The EBOV glycoprotein (GP) gene encodes severaltranscripts due to RNA editing at a conserved site consisting of 7 consecutiveuridines. The majority of GP gene transcripts are unedited and encodefor secreted sGP, whereas surface GP is synthesized from edited mRNAscontaining an extra adenosine inserted at the editing site by viral polymerase.Here we demonstrate for the first time that the editing site can serve asa cryptic transcriptional stop signal causing synthesis of truncated mRNA.Northern blot analysis revealed that nearly 50% of GP mRNAs in EBOVinfected cells represent full length GP transcripts whereas others are productsof premature transcription termination. Sequence analysis of shortmRNAs showed a presence of poly(A) tail consisting of 27 44 adenosinesand an absence of translation termination codon at the end of readingframe. Expression of this nonstop mRNA was assayed using a truncatedpoly adenylated mRNA (tr mRNA) synthesized in vitro from a plasmid byT7 polymerase. No specific protein was detected when capped tr mRNAwas used for transfection of 293T cells whereas its in vitro translationresulted in synthesis of a 36kDa GP specific polypeptide. Of note, introductionof stop codon upstream to the poly(A) sequence restored proteinsynthesis indicating that mammalian cells possess a mechanism preventingexpression of aberrant mRNAs. Role and significance of the editingsite as well as function of novel GP gene product in replication of EBOVwill be discussed.REF 237Hepatocyte pathway alterations in response to in vitro Crimean CongoHemorrhagic Fever virus infectionChristophe FRAISIER 1,2 , Raquel RODRIGUES 3 , Vinh VU HAI 1,2 , LucCAMOIN 4,5 , Maya BELGHAZI 6 , Stéphanie BOURDON 1 , PatrickFOURQUET 4,5 , Glaucia PARANHOS BACCALA 3 , LionelALMERAS 1,2 , Christophe Nicolas PEYREFITTE 3,71 Unité de Parasitologie, Institut de Recherche Biomédicale des Armées(IRBA) antenne Marseille, Parc du Pharo, Marseille, FRANCE; 2 Unitéde Recherche des Maladies Infectieuses et Tropicales Emergente(URMITE), Marseille, FRANCE; 3 Emerging Pathogens Laboratory, FondationMérieux, Lyon, FRANCE; 4 Inserm, U1068, CNRS, UMR7258,CRCM, Aix Marseille Univ, UM 105, Marseille, FRANCE; 5 Institut PaoliCalmettes, Marseille Protéomique, Marseille, FRANCE; 6 Aix MarseilleUniversité, CNRS, UMR 7286, Marseille, FRANCE; 7 Unité de virologie,Institut de Recherche Biomédicale des Armées (IRBA) antenne de Lyon,tour CERVI, Lyon, FRANCECrimean Congo hemorrhagic fever virus (CCHFV) is tick borne virus responsiblefor hemorrhagic manifestations and multiple organ failure, witha high mortality rate. In infected humans, damage to endothelial cells andVirologie, Vol 17, supplément 2, septembre 2013S185