rologie i - European Congress of Virology

5 th European Congress of Virologyrange from asymptomatic to hemorrhagic fevers, encephalitis and/or hepatitiswith high fatality rates in severe cases. Approved vaccines andefficient antiviral treatments are unavailable. Our work focuses on thecell entry of Bunyaviruses and we use Uukuniemi virus (UUKV, genusPhlebovirus) as a model system. Target cell penetration by UUKV islow pH dependent and believed to occur from within a late endosomalcompartment (Lozach et al. 2010 and 2011), presumably by membranefusion. To analyze membrane fusion in more detail, we developed anin vitro fusion assay using fluorescent UUKV particles and/or fluorescentliposomes. We show that low pH mediated UUKV/liposome lipidmixing and liposome content release is favoured by negatively chargedlipid head groups in target membranes. To complement the biophysicalassays, we use electron cryo tomography to study intermediate structuresarising during UUKV fusion. By combining functional and structural data,we wish to unravel mechanistic details of UUKV fusion. This will lead toa more detailed understanding of Bunyavirus entry and thus a more solidfoundation for vaccine and drug development.H stalks “opens” to trigger F, last models suggest that tetrameric H headsmay also move as “dimeric blocs” to transmit signals for F activation. Wecharacterized an anti CDV H mAb (1347), which strongly inhibited cellcell fusion. Epitope mapping, using soluble H forms, revealed a segmentin the H stalk, which, once transposed into a foreign protein, allowed forits recognition by 1347. 1347 did not cross react with MeV H, but substitutionof two residues in the epitope (by the corresponding ones in CDVH) enabled MeV H detection and cell cell fusion inhibition. Importantly,the epitope locates above the putative F contacting zone, which correlatedwith 1347 binding activity being unaffected by the presence of F. However,1347 bound to a membrane bound “headless” CDV H variant with a higherefficiency, suggesting that H heads, in a pre receptor bound state, impededfull 1347 recognition. Remarkably, a spontaneous basal level of F triggeringwas monitored in headless H/F coexpressing cells, and this, regardlessof the presence or absence of 1347. Overall, these findings suggest that1347 does not inhibit F activation by impairing H stalks dependent intrinsictriggering activity. Rather, we suggest that 1347 may alter putativereceptor induced H heads to stalks “un clamping” movements.REF 151Murine hepatitis coronavirus is a late penetrating virus that requiresa functional HOPS complex for fusionCornelis DE HAAN, Christine BURKARD, Hélène VERHEIJE, OliverWICHT, Peter ROTTIER, Berend Jan BOSCHUtrecht University, Utrecht, THE NETHERLANDSMany viruses that are taken up via endocytosis depend on endosomal maturationand the associated pH drop to induce virus cell fusion. Particularlylate penetrating viruses have been described as sensitive to perturbations ofendosomal maturation. Murine hepatitis virus (MHV), a prototypic coronavirus,contains furin cleaved spike fusion proteins. It does not appear torequire a low pH for activation of its fusion protein and is able to inducecell cell fusion. While these observations are indicative of cell entry byfusion at the plasma membrane, we and others have recently also shownthat MHV relies on clathrin mediated endocytosis for productive entry.To solve this apparent contradiction we have studied the entry pathway ofMHV in more detail using specific drugs, siRNAs and knock out cells incombination with virus replication dependent reporter and novel replicationindependent fusion assays. Our results confirm the important role ofclathrin mediated endocytosis and indicate the involvement of the actincytoskeleton in virus entry. In addition, MHV was shown to be criticallydependent on endosomal maturation for successful infection. Virions colocalizedwith Rab7 and LAMP1, markers of late endo lysosomal vesicles,while virus cell fusion was impaired in cells lacking Rab7 or a functionalHOPS complex. These results indicate that MHV, despite carrying a furincleaved fusion protein that induces cell cell fusion, is a late penetratingvirus that needs to be transported to the late endo lysosomal compartmentsfor virus cell fusion and productive entry to occur.REF 152Inhibition of Membrane Fusion by a mAb interacting with theMorbillivirus Attachment Protein Stalk DomainNadine EBERT ADER 1,2 , Philippe PLATTET 1 , AndreasZURBRIGGEN 11 Division of Neurological Sciences, DCR VPH, Vetsuisse faculty, Universityof Bern, Bern, SWITZERLAND; 2 Graduate School for Cellular andBiomedical Sciences, University of Bern, Bern, SWITZERLANDMeV and CDV entry systems rely on two envelope glycoproteins: theattachment (H) and the fusion (F) proteins that co operate to achieve plasmamembrane fusion. The H ectodomain consists of a stalk supporting a headthat contacts different receptors. While the central section of morbillivirusREF 153Characterization of the core structure of glycoprotein B of Herpessimplex virus 1Massimiliano GALDIERO 1,3 , Marco CANTISANI 2,3 , AnnaritaFALANGA 2 , Novella INCORONATO 1 , Luigi RUSSO 1 , Alfonso DESIMONE 4 , Rita BERISIO 5 , Stefania GALDIERO 2,3,51 Department of Experimental Medicine II University of Naples, Napoli,ITALY; 2 Department of Pharmacy University of Naples Federico II,Napoli, ITALY; 3 Centro Interuniversitario di Ricerca sui Peptidi Bioattivi,University of Naples Federico II, Napoli, ITALY; 4 Division of MolecularBiosciences, Imperial College, London, UNITED KINGDOM; 5 Istituto diBiostrutture e Bioimmagini, CNR, Napoli, ITALYEntry of enveloped viruses requires fusion of viral and cellular membranes,driven by conformational changes of viral glycoproteins. The crystallizedtrimeric form of glycoprotein B of Herpes simplex virus has been describedas a post fusion conformation and several studies prove that as other classIII fusion proteins gB undergoes a pH dependent switch between the preand post fusion conformation. Here, using peptides corresponding to thelong helix of the post fusion structure and several biophysical techniques,including fluorescence and circular dichroism spectroscopies, surfaceplasmon resonance, and molecular dynamic simulations, we provideevidence of the existence of a thoroughly different pre fusion conformation;during the conformational rearrangements, some epitopes are lost orformed, which supports the hypothesis for the search of a minimal peptidicsequence able to interact with the virus in its prefusogenic conformation.REF 154Receptor use of Lassa virus in human dendritic cellsAna Rita GONÇALVES 1 , Marie Laurence MORAZ 1 , AntonellaPASQUATO 1 , Ari HELENIUS 2 , Pierre Yves LOZACH 2 , Stefan KUNZ 11 Institute of Microbiology, Lausanne University Hospital and University ofLausanne, Lausanne, SWITZERLAND; 2 Institute of Biochemistry, FederalInstitute of Technology Zurich, Zurich, SWITZERLANDThe arenavirus Lassa (LASV) causes a severe hemorrhagic fever with highmortality in humans. Antigen presenting cells, in particular dendritic cells(DC), are early and preferred targets of LASV and their productive infectioncontributes to virus induced immunosuppression observed in fataldisease. Here we characterized the role of LASV candidate receptors inviral entry into primary human DCs using a chimera of the prototypicarenavirus lymphocytic choriomeningitis virus (LCMV) expressing theVirologie, Vol 17, supplément 2, septembre 2013S161