rologie i - European Congress of Virology

5 th European Congress of VirologyFriday 13 th September 2013, 8h30 – 10h30WORKSHOP 10: “VIRUS ENTRY: ENVELOPEGLYCOPROTEINS, RECEPTORS, ENDOCYTOSIS”KEYNOTE:Chairpersons: Niklas ARNBERG (Umea, SWEDEN)& Urs GREBER (Zürich, SWITZERLAND)Room Gratte-Ciel 1, 2, 3Intermediate structures in the fusion associated transition of vesiculovirusglycoproteinEduard BAQUERO 1 , Aurélie A. ALBERTINI 1 , Malika OULDALI 1 ,Linda BUONOCORE 2 , John K. ROSE 2 , Jean LEPAULT 1 , StéphaneBRESSANELLI 1 , Yves GAUDIN 11 Laboratoire de Virologie Moléculaire et Structurale, CNRS (UPR 3296),Avenue de la Terrasse, 91198, Gif sur Yvette Cedex, FRANCE; 2 YaleUniversity School of Medicine, 310 Cedar St., New Haven, CT 06510,USAEnveloped viruses enter cells through a membrane fusion reaction drivenby conformational changes of viral fusion glycoproteins. Three differentclasses of viral fusion proteins have been identified to date based on theircommon structural motifs. Crystal structures have provided atomic, staticpictures of pre- and post-fusion conformations for several of these glycoproteins;however, no atomic structure of a transitional intermediate thatcould form the bridge between the viral and cellular membranes is known.VSV G is the prototype of the third class and forms different trimers inits pre- and post-fusion conformations. We report here a crystal structurefor G of Chandipura virus, another vesiculovirus responsible fordeadly encephalopathies. In this single crystal, two distinct conformationscorresponding to early and late refolding states of G, arranged in aflat tetrameric assembly exposing fusion loops. Consistent with these data,electron microscopy and tomography show different intermediates at theviral surface depending on experimental conditions.This work reveals the chronological order of the structural changes in theprotein and offers a detailed pathway for the conformational transition.Particularly, our data confirm that the conformational change involvesmonomeric intermediates and that it likely proceeds to an elongated hairpinmonomer before subsequent collapse into the post-fusion trimer.Furthermore, our data and previously published mutagenesis analysis indicatethat after dissociation of the pre-fusion trimer into monomers, vesiculovirusfusion glycoprotein could re-associate not only into trimers but alsointo a dimeric (and even tetrameric) assembly which is optimally organizedto forming the initial bridge between the target and viral membranes.ORAL COMUNICATIONSREF O83Dipeptidyl peptidase 4: Entry portal for the emerging human coronavirusEMCBerend Jan BOSCH 1 , Huihui MOU 1 , Stalin RAJ 2 , Saskia SMITS 2,7 ,Dick DEKKERS 3 , Marcel MÜLLER 4 , Ronald DIJKMAN 6 , JeroenDEMMERS 3 , Ali ZAKI 5 , Ron FOUCHIER 2 , Volker THIEL 6,8 ,Christian DROSTEN 4, Albert OSTERHAUS 2 , Peter ROTTIER 1 , BartHAAGMANS 21 Virology Division, Department of Infectious Diseases & Immunology,Faculty of Veterinary Medicine, Utrecht University, Utrecht, THENETHERLANDS; 2 Viroscience Lab, Erasmus Medical Center, Rotterdam,THE NETHERLANDS; 3 Proteomics Department, Erasmus Medical Center,Rotterdam, THE NETHERLANDS; 4 Institute of Virology, Universityof Bonn Medical Centre, Bonn, GERMANY; 5 Virology Laboratory, DrSoliman Fakeeh Hospital, Jeddah, KINGDOM OF SAUDI ARABIA;6 Institute of Immunobiology, Kantonal Hospital St. Gallen, St. Gallen,SWITZERLAND; 7 Viroclinics Biosciences BV, Rotterdam, THE NETHER-LANDS; 8 Vetsuisse Faculty, University of Zürich, Zürich, SWITZERLANDCoronaviruses generally cause respiratory and enteric infections and arefound in many mammalian and avian species. They have a marked potentialfor host species switching by adapting to receptors of novel host species.Recently a novel coronavirus (hCoV EMC) was identified in patients withsevere and sometimes lethal lower respiratory tract infection. The emergingvirus is most closely related to CoVs found in bats. We recently haveidentified dipeptidyl peptidase 4 (DPP4) - also known as CD26 - as a functionalreceptor for hCoV EMC. DPP4, an exopeptidase found to occur onhuman lung surfaces, specifically co purified with the receptor binding S1subunit of the hCoV EMC spike protein from lysates of susceptible Huh7 cells. Antibodies directed against DPP4 inhibited hCoV EMC infectionof primary human bronchial epithelial cells and Huh 7 cells. We showthat non susceptible cells become infectable upon transient expression ofthe DPP4 receptor. Moreover, we found that the virus was able to use theevolutionary conserved DPP4 of other species, most notably bats. The peptidaseactivity of DPP4 was not necessary for virus entry. Furthermore wedefined the interacting domains within the S1 subunit as well as the receptor.Ongoing studies focus on the spike promiscuity for DPP4 proteins ofother species and the DPP4 receptor usage by related bat coronaviruses.The discovery of DPP4 as a functional receptor will help us understand thepathogenesis and epidemiology of this emerging virus and may facilitatethe development of intervention strategies.REF O84Human hepatitis B and D Viruses exploit sodium taurocholate cotransporting polypeptide (NTCP) to bind and enter hepatocytes ina species specific mannerYi NI 1 , Florian LEMPP 1 , Stefan MEHRLE 1 , Holger SÜLTMANN 2 ,Stephan URBAN 11 Department of Infectious Diseases, Molecular Virology, University HospitalHeidelberg, Heidelberg, GERMANY; 2 Cancer Genome Research(B063), Deutsches Krebsforschungszentrum/NCT, Heidelberg, GER-MANYHepatitis B (HBV) and Hepatitis D virus (HDV) binding to a hepatic receptorrequires a myristoylated N terminal preS domain of the large HBVenvelope protein. Accordingly, a synthetic lipopeptide comprising the respectivepreS sequence (Myrcludex B) specifically binds this receptor andblocks infection. In vivo pharmacokinetic studies of Myrcludex B in severalspecies revealed a hepatocyte specific receptor expression in even nonHBV susceptible hosts (e.g. mouse). Using a screening approach we identifiedsodium taurocholate cotransporting polypeptide (NTCP) as a possiblecellular target of Myrcludex B. In a biochemical approach this moleculehas also been identified recently as a functional HBV/HDV receptor.We here demonstrate that constitutive expression of mouse (m) or human(h) NTCP in human (HepG2, HuH 7, HepaRG cells) and mouse hepaticcells lines (Hepa1 6, Hep56D) render them competent for binding withMyrcludex B. However, hNTCP but not mNTCP expressing cell lines ofboth species became susceptible to HDV infection, indicating that mNTCPalthough competent in preS binding cannot support complete virus entry.Using a set of chimeras of hNTCP and mNTCP we mapped an extracellularloop domain which determines the susceptibility. In contrast to HDV, HBVinfection is completely abolished in the mouse cell lines, suggesting a hostspecific restriction during HBV infection.In conclusion we demonstrate the crucial role of NTCP as the specificHBV/HDV receptor and mapped an essential NTCP domain required forhost specificity.S72 Virologie, Vol 17, supplément 2, septembre 2013