rologie i - European Congress of Virology

5 th European Congress of VirologyThursday 12 th September 2013, 8h30 – 10h30WORKSHOP 12: “VIRAL GENE EXPRESSION –TRANSCRIPTION, TRANSLATION”Chairpersons:Richard ELLIOTT (Glasgow, UNITED KINGDOM)& Peter ROTTIER (Utrecht, THE NETHERLANDS)Room Tête d’OrThis RNA motif presented high homology in sequence and secondarystructure with the translation inhibitor GAIT element that is present at the3 ′ end of several mRNAs coding proinflammatory proteins. In response toIFN-, the GAIT complex (EPRS, hnRNP Q, L13a, and GAPDH) interactswith the GAIT element and suppresses the translation. Similarly tothe cellular GAIT element, the presence of the viral motif at the 3 ′ UTR ofan mRNA encoding the luciferase gene inhibited its translation in the presenceof the GAIT complex, suggesting that the viral GAIT motif could beinvolved on translation silencing. It could be postulated that the viral GAITcould act either promoting the viral genome translation silencing duringthe translation replication switch or interfering with the host inflammatoryresponse.KEYNOTE:Norovirus translation: a novel paradigm for viral protein synthesisIan GOODFELLOWUniversity of Cambridge, UNITED KINGDOMNoroviruses are now recognized as the major cause of viral gastroenteritisin the developed world. Despite this impact, our understanding of the biologyof these viruses lags significantly behind that of other RNA viruses.Human noroviruses remain unable to replicate in the currently availablecell culture systems. Murine norovirus is currently used as model for elucidatingmolecular details of noroviruses replication due to the fact thatit can be propagated in cell culture, has reverse genetics systems and anavailable animal model.Noroviruses and other members of the Caliciviridae use a novel mechanismof viral genome translation not yet observed in any other animalRNA virus. This mechanism relies on the interaction of cellular translationinitiation factors with a virus-encoded protein covalently linked tothe viral RNA genome, VPg. In collaboration with Stephen Curry andStephen Mathews (Imperial College) we have determined the structure ofthe norovirus VPg protein. Mutational analysis has then been used to identifyfunctional sites within VPg and to identify those residues importantfor the interaction with initiation factors. Further insights into the role ofVPg in viral translation have been obtained from proteomic analysis ofhost factors associated with VPg. To further understand the effect of virusinfection on the host cell translation initiation apparatus we have begunto use quantitative proteomic approaches and a number of other methodsto understand the effect of norovirus infection on the host cell translationapparatus.ORAL COMMUNICATIONSREF O31Identification of an RNA translational regulator element at the 3 ′ endof the TGEV genomeFernando ALMAZAN, Silvia MARQUEZ JURADO, Nogales AITOR,Enjuanes LUISDepartment of Molecular and Cell Biology, National Center of Biotechnology(CNB CSIC), Darwin 3, Madrid, SPAINCoronavirus (CoV) replication and transcription are complex processesthat require the specific recognition of RNA cis acting signals located atthe ends of the viral genome. To identify cell proteins involved in CoVreplication, transmissible gastroenteritis CoV (TGEV) genome ends wereused as baits for RNA affinity chromatography purification. Nine proteinspreferentially bound to the 3 ′ end were identified, from which, a functionalrole on CoV RNA synthesis was demonstrated for hnRNP Q, glutamyl prolyltRNA synthetase (EPRS), arginyl tRNA synthetase (RRS), and poly(A)binding protein (PABP). The RNA motifs interacting with PABP, EPRSand RRS were identified. The PABP bound to the poly(A), while the EPRSand RRS bound to a 32 nt motif located at 411 nt from the 3 ′ genome end.REF O32TIA 1 is a cellular restriction factor for tick borne encephalitis virusthat binds viral RNA and is recruited to perinuclear sites of viralreplicationAlessandro MARCELLO, Amelina ALBORNOZ, Lisa MIORINICGEB, Trieste, ITALYTick borne encephalitis virus (TBEV) replication compartments are formedof 80 nm vesicles where the viral genomes are replicated and aconfined extravesicular space where replicated viral RNA is retained fortranslation and assembly (Miorin et al. J Virol 2013). These structurescontribute to the early delay in interferon signalling observed in infectedcells (Miorin et al. Virus Res 2012). In this work we investigated thehost cell factor T intracellular antigen 1 (TIA 1), a stress induced RNAbinding protein involved in the repression of initiation of translation ofcellular mRNAs through the formation of stress granules (SG). TIA 1interacts with viral RNA as shown by RNA immuneprecipitation of TIA1 in TBEV infected cells. During TBEV infection, cytoplasmic TIA 1 isrecruited at sites of viral replication with concomitant depletion from SGs.This effect is TIA 1 specific since G3BP1, another SG protein, remainsin SGs and doesn’t re localize to sites of viral replication. Moreover, heatshock induction of TIA 1 SGs, but not G3BP1 SGs, is inhibited in TBEVinfected cells. TIA 1 depletion leads to an increase of TBEV RNA levelsas well as extracellular infectivity. Taking advantage of a TBE luciferasereplicon system we could observe increased signal also at early timepoints consistent with TIA 1 mediated inhibition at the level of translation.These results indicate that TIA 1 is a cellular restriction factor duringTBEV replication that binds viral RNA and is recruited to extravesicularsites of replicated viral RNA.REF O33Structure of the full length hepatitis C virus IRES in solutionMarc JAMIN 1 , Julien PÉRARD 1 , Cédric LEYRAT 1 , FlorenceBAUDIN 1,2 , Emmanuel DROUET 11 UMI3265 UJF EMBL CNRS UVHCI, Grenoble, FRANCE; 2 EuropeanMolecular Biology Laboratory (EMBL), Heidelberg, ALLEMAGNEThe 5 ′ untranslated region of the hepatitis C virus genome contains aninternal ribosome entry site that initiates cap independent translation ofthe viral RNA. Atomic structures of different fragments of this large RNAregion have been determined but, until now, the structural characterizationof the entire internal ribosome entry site remained limited to cryo electronmicroscopy reconstructions of the internal ribosome entry site bound to differentcellular partners. Here we report an atomic model of free full lengthhepatitis C virus internal ribosome entry site refined by selection againstsmall angle X ray scattering data that incorporates the known structures ofdifferent fragments. We found that an ensemble of conformers reproducessmall angle X ray scattering data better than a single structure suggestingin combination with molecular dynamics simulations that hepatitis Cvirus internal ribosome entry site is an articulated molecule made of rigidVirologie, Vol 17, supplément 2, septembre 2013S49