rologie i - European Congress of Virology

5 th European Congress of VirologyFriday 13 th September 2013, 15H00 – 17h15WORKSHOP 7: “CELLULAR FACTORS CONTROLINGVIRAL INFECTION AND ROLE OF HOST GENETICS”Chairpersons: Frank KIRCHHOFF (Ulm, GERMANY)& Aine McKNIGHT (London, UNITED KINGDOM)Room Prestige Gratte-CielKEYNOTE:Host cell factors and pathways promoting and restricting hepatitis Cvirus replicationRalf BARTENSCHLAGERDepartment of Infectious Diseases, Molecular Virology, University of Heidelberg,Im Neuenheimer Feld 345, Heidelberg, GERMANYHepatitis C viruses (HCV) comprise a group of positive-strand RNAviruses belonging to the Flaviviridae family. As a major cause of acuteand chronic liver disease worldwide, HCV has received much attention.With the advent of highly efficient and robust cell culture systems forHCV, the principles of the viral replication cycle have been unravelled. Asurprisingly high number of molecules that are essential for or that promoteHCV entry into hepatocytes has been identified. Moreover, importantinsights into the biogenesis and architecture of the membranous replicationcompartment induced upon viral infection have been gained. Severalhost cell factors required for formation or activity of the viral replicationmachinery, such as cyclophilin A and phosphatidyl inositol-4-kinase IIIalpha,have been identified and they represent attractive targets for hostfactor-targeting antiviral therapy. Finally, it was found that HCV assemblyoccurs in close association with cytosolic and luminal lipid dropletsexplaining, at least in part, why HCV particles are tightly associated withhost cell lipoproteins and lipids.A hallmark of HCV infection is the high rate of persistence (∼80%)arguing that this virus has developed efficient strategies to overcome innateand adaptive immune responses. It is well established that the NS3 serinetypeprotease blocks the induction of antiviral cytokines by proteolyticcleavage of adaptor proteins involved in RIG-I and TLR-3 dependentsignalling. Paradoxically however, in infected patients interferon-inducedgenes are activated and the degree of activation negatively correlates withoutcome of interferon-based therapy. While the underlying mechanismsremain to be determined, it is obvious that HCV has developed strategiesto sustain persistence in spite of an antiviral program.ORAL COMMUNICATIONSREF O101Hepatitis C virus infection modulates the central carbon metabolismof the cellOlivier DIAZ 1,2 , Christophe RAMIÈRE 1,2,3 , Jonathan RODRIGUEZ 1,2 ,Aurélie BADILLO 4 , Jean Charles PORTAIS 5 , Vincent LOTTEAU 1,2 ,Patrice ANDRÉ 1,2,31 Université de Lyon, Lyon, FRANCE; 2 INSERM U1111 – CNRSUMR5308 Université Lyon 1, ENS de Lyon, Lyon, FRANCE; 3 HospicesCivils de Lyon, Lyon, FRANCE; 4 UMR 5086 CNRS Université Lyon 1,Lyon, FRANCE; 5 UMR5504, UMR792 Ingénierie des Systèmes Biologiqueset des Procédés CNRS, INRA, INSA, Toulouse, FRANCEPerturbations of glucido lipidic metabolism, induced by Hepatitis C virus(HCV) infection in the hepatocyte, are not completely elucidated. Recentlyseveral viruses were described as capable to subvert central carbon metabolism(CCM) to support their replication and assembly. We thus investigatedhow HCV modulates CCM. We monitored uptake and release of key metabolitesin supernatants of HCV infected Huh7.5 cells during 72 h postinfection. Glucose and glutamine uptake and lactate excretion were increasedindicating glycolysis modulations in infected cells. We identified theprotein protein interaction of NS5A with hexokinase (HK), the first andrate limiting enzyme of glycolysis. Enzymatic HK assay performed withcytosolic fraction of cells replicating the HCV subgenomic replicon (Huh913) revealed an activity increase. The same observation was performed incells overexpressing only NS5A. Moreover, addition of purified NS5A inin vitro HK assay was sufficient to increase HK activity. To further definefunctionally how HCV modulate CCM, 42 different metabolic moleculesreferred as intermediates or connected to glycolysis and citrate cycle metabolismwere analyzed in HCV infected Huh7.5 cells using HPAEC andIDMS. Data revealed that beside the apparent global increased glycolysisin infected cells, nucleotide precursor’s synthesis appeared enhancedwhereas no changes were observed within cell energy status. All togetherthese observations indicate a direct interaction between non structuralHCV proteins and glycolysis enzymes at the origin of CCM modulation byHCV.REF O102Coxsackievirus Hijacks Host miRNA 126 to Mediate Cross talkof ERK and Wnt/beta catenin Signal Pathways for Its ReplicationDecheng YANG, Xin YE, Maged HEMIDA, Ye QIU, Paul HANSON,Huifang Mary ZHANGUniversity of British Columbia, Vancouver, CANADACoxsackievirus B3 (CVB3), a positive single stranded RNA virus, is amember of the Enterovirus genus in the Picornaviridae family. It is the primarypathogen of viral myocarditis. CVB3 infection causes directed injuryof the heart by rapid replication in the myocardium. However, the molecularpathways leading to myocarditis remain unclear. microRNA (miRNA),as a novel regulator of gene expression, may play an essential role in CVB3pathogenesis. To study the functional role of miRNAs in modulating CVB3replication, we selected a heart abundant miRNA, miR 126, to test ourhypothesis. Here we describe a novel mechanism by which miR 126 regulatestwo signal pathways essential for CVB3 replication. We found thatCVB3 induced ERK1/2 activation triggered the phosphorylation of ETS1/2 transcription factors, which induced miR 126 upregulation. By usingboth miR 126 mimics and its inhibitors, we proved that the upregulatedmiR 126 suppressed SPRED1(sprout related, EVH1 domain containing 1)expression and in turn enhanced ERK1/2 activation. This positive feedbackloop of ERK1/2 miR 126 ERK1/2 promoted CVB3 replication at earlyphase of infection. Meanwhile, miR 126 expression stimulated the GSK3b activity and induced degradation of beta catenin via suppressing LRP6(low density lipoprotein receptor related protein 6) and WRCH1(Wnt responsiveCdc42 homolog 1), two newly identified targets in the Wnt/catenin pathway, which sensitized the cells to virus induced cell deathand increased viral progeny release to initiate new infections. Our resultsdemonstrate for the first time that dynamically upregulated miR 126 uponCVB3 infection targets SPRED1, LRP6 and WRCH1 genes, mediatingcross talk between ERK1/2 and Wnt/beta catenin signal pathways, andthus promotes viral replication and contributes to the viral cytopathogenicity.Virologie, Vol 17, supplément 2, septembre 2013S83