rologie i - European Congress of Virology

5 th European Congress of VirologyREF O85LDL Receptor and its family members are the cellular receptors ofVSV and VSV G pseudotyped viral vectorsMenachem RUBINSTEIN, Danit FINKELSHTEIN, Daniela NOVICK,Sara BARAKWeizmann Istitue of Science, Rehovot, ISRAELVesicular stomatitis virus (VSV) exhibits a remarkably robust and pantropicinfectivity, mediated by its coat protein, VSV G. Utilizing this property,recombinant forms of VSV and VSV G pseudotyped viral vectors arebeing developed for gene therapy, vaccination and viral oncolysis, and areextensively employed for gene transduction in vivo and in vitro. The broadtropism of VSV suggests that it enters cells through a highly ubiquitousreceptor whose identity has so far remained elusive. Here, we show thatthe low density lipoprotein receptor (LDLR) serves as the major entryport of VSV and of VSV G pseudotyped lentiviral vectors in human andmouse cells, whereas other LDLR family members serve as alternativereceptors. The widespread expression of LDLR family members accountsfor the pantropism of VSV, and for the broad applicability of VSV Gpseudotyped viral vectors for gene transduction. (PNAS, 2013, in press)REF O86Changes in SV40 binding to its receptor GM1 affects vacuolizationand plaque formation in CV1 monkey cellsNasim MOTAMEDI 1 , Thomas G. MAGALDI 1,2 , Xiaochu MA 1 , DanielDIMAIO 1,4,51 Department of Genetics, Yale School of Medicine, New Haven, CT, USA;2 Tumor Virus Molecular Biology Section, Laboratory of Cellular Oncology,National Cancer Institute, Bethesda, MD, USA; 3 Department ofTherapeutic Radiology, Yale School of Medicine, New Haven, CT, USA;4 Yale Cancer Center, Yale School of Medicine, New Haven, CT, USA;5 Department of Molecular Biophysics and Biochemistry, Yale School ofMedicine, New Haven, CT, USAPolyomaviruses are small non enveloped DNA viruses with the capsid surfacecomposed entirely of the structural protein VP1. Polyomavirus VP1 sbind to oligosaccharide residues on their cell surface receptors. Simianvirus 40 (SV40), a monkey polyomavirus, binds to the terminal sialic acidresidue of the ganglioside GM1 to successfully infect cells. A hallmark ofSV40 infection in monkey cells is the dramatic vacuolization of cells priorto lysis and the subsequent formation of plaques in cultured cells. We haveintroduced mutations in the GM1 binding site of VP1 that inhibit SV40 bindingto GM1. However, these mutant viruses can still infect monkey cells,albeit at a lower efficiency. Interestingly, these mutant viruses fail to inducethe formation of cellular vacuoles or form plaques. Vacuolization is alsoinhibited by modulation of GM1 levels by siRNA knockdown of enzymesinvolved in ganglioside synthesis. Introducing additional mutations intothe GM1 binding site of VP1 rescues the phenotype of defective vacuolizationand plaque formation in monkey cells without restoring capsidbinding to GM1. These mutants presumably bind to a different oligosaccharide.Taken together, these experiments indicate that the ability of VP1to bind gangliosides, most likely GM1, is essential for plaque formationand vacuolization in CV1 cells and that gangliosides are important forstages of infection in addition to cell entry.REF O87Identification of functional networks in E1E2 glycoproteins unveilsHepatitis C virus fusion mechanisms and new therapeutic opportunitiesFlorian DOUAM 1,2 , Linda DIB 3,4 , Viet Loan DAO THI 1 , GuillemetteMAURIN 1 , Loic SCHWALLER 1,5 , Judith FRESQUET 1 , DimitriMOMPELAT 1 , François Loic COSSET 1 , Alessandra CARBONE 3 ,Dimitri LAVILLETTE 1,21 CIRI, International Center for Infectiology Research, Inserm U1111,Ecole Normale Supérieure de Lyon, Université Lyon 1, CNRS, Lyon,FRANCE; 2 Microbial Dynamics and Viral Transmission,CNRS, UMR5557 Ecologie Microbienne, Université Lyon 1, Villeurbanne, FRANCE;3 Laboratoire de Génomique des Microorganismes, UMR7238 CNRSUPMC, Paris, FRANCE; 4 Molecular Phylogenetics and Speciation,Département d’écologie et évolution, Université de Lausanne, Lausanne,SUISSE; 5 Statistique & Génome, AgroParisTech, INRA MIA, UMR 518,Paris, FRANCEChronic Hepatitis C virus (HCV) infection is a health threat affectingmore than 130 million people worldwide. Since cell entry is the firststep of virus host interaction, it represents a promising target for antiviraltherapies. However, the HCV entry process at the molecular levelis still poorly understood. Important domains of HCV E1 and E2 glycoproteinsinvolved in this process remain to be discovered and especiallytheir inter relationship during the entry process must be defined. In orderto identify such domains and interactions, we used two complementarystrategies: on the one hand, a classical molecular virology approach basedon the functional comparison of properties of two different strains, andon the other hand, a structural modeling and a combinatorial analysisof many strains of different genotypes helped to reconstitute networksof coevolving residues within and between viral glycoproteins. Differentchimeras and point mutants were analyzed in assays addressing assembly,binding and fusion using both, pseudotyped retroviral particles andcell cultured derived viruses. By these approaches, we were able to identifyparticular dialogs in different genotypes between residues in E1 andthe domain III of E2 which are critical for membrane fusion. Our resultshighlight that HCV has developed an original fusion mechanism, likelyconcealing some properties of both flavi and pesti virus fusion processes.We are currently developing strategies to exploit the potential therapeuticopportunity represented by critical E1E2 conformational changes duringentry.REF O88Targeted antiviral siRNA screen identifies the poxvirus genome uncoatingfactorSamuel KILCHER, Florian Ingo SCHMIDT, Christoph SCHNEIDER,Manfred KOPF, Ari HELENIUS, Jason MERCER1 Institute of Biochemistry ETH Zürich, Zürich, SWITZERLAND; 2 Instituteof Molecular Biomedicine ETH Zürich, Zürich, SWITZERLANDSince its discovery, RNA interference (RNAi) has been established asthe method of choice for large scale loss of function studies. Host factorrequirements of a variety of pathogens have been addressed using genomewide siRNA screens. In addition, viral genomes or mRNAs have beenVirologie, Vol 17, supplément 2, septembre 2013S73