rologie i - European Congress of Virology

5 th European Congress of VirologyLASV glycoprotein (rLCMV LASVGP). Several cellular receptors havebeen described for LASV, including the ECM receptor dystroglycan (DG),the Tyro3/Axl/Mer (TAM) receptors Axl and Tyro3, and the C type lectinDC specific ICAM 3 grabbing nonintegrin (DC SIGN). We found thatprimary human monocytes and monocyte derived DCs (MDDC) expressonly low levels of functional DG and lack Axl and Dtk. Differentiationof monocytes into DCs enhanced virus attachment and entry, concomitantwith the up regulation of DC SIGN. The binding of LASV to DC SIGNwas mediated by mannose sugars located on the N terminal GP1 subunitof LASVGP. DC SIGN served as an attachment factor for LASV andaccelerated capturing of free virus by MDDCs, facilitating productiveinfection. However, in contrast to the Phlebovirus Uukuniemi virus, knownto use DC SIGN as an authentic entry receptor in DCs, infection withrLCMV LASVGP was less dependent on DC SIGN and showed strikinglydistinct kinetics. The data at hand provide evidence for a role of DCSIGN as an enhancing factor for LASV entry into human DCs and suggestthe existence of other yet unknown receptor(s) involved in productiveLASV infection in this crucial cell type.This research was supported by Swiss National Science Foundation grantFN 310030 132844 (S.K.).REF 155Characterization of binding properties of the IBV spike proteinMartina HESSE, Georg HERRLER, Christine WINTERInstitute for Virology, Hannover, GERMANYThe Infectious Bronchitis Virus (IBV) belongs to the family of Coronaviridae,which comprises a variety of animal and human pathogens. A crucialstep in the viral lifecycle is the entry into host cells, which begins withviral attachment. For IBV it is known that attachment is mediated by theS1 subunit of the spike protein. a2,3 linked sialic acids serve as its receptordeterminants, but the exact localization of the sialic acid binding domainwithin the S1 subunit is still not identified. It is the aim of our study tofurther analyse the binding characteristics of the IBV spike protein. As atool for our investigations we use soluble chimeric proteins which consistof the S1 subunit of different IBV spike proteins fused to a human IgG Fctag. The constructs allow analysing the attachment of spike proteins to primarychicken cells and tissues in different settings by detecting the Fc tagwith fluorochrome labelled antibodies. For identification of the IBV sialicacid binding site certain domains of the spike protein have been deletedand the mutant’s binding ability was addressed. First experiments revealedthat a deletion of 42 aminoacids at the N Terminus of the spike proteinreduces the binding to host cells clearly. Mutants with smaller deletionscould narrow the responsible site down to seven amino acids. An alaninscan is performed to identify the aminoacids important for binding. Toconfirm that the respective domain is responsible for sialic acid binding itis cloned into Coronavirus spike proteins which do not possess sialic acidbinding properties of their own.REF 156Molecular Basis of Canine Distemper Virus Cell Entry through theHuman CD150/SLAM ReceptorMojtaba KHOSRAVI 1,2 , Lisa ALVES 1,2 , Maria BIERINGER 3 , JürgenSCHNEIDER SCHAULIES 3 , Andreas ZURBRIGGEN 1 , PhilippePLATTET 11 Division of Neurological Sciences, DCR VPH, Vetsuisse faculty, Universityof Bern, Bern, SWITZERLAND; 2 Graduate School for Cellularand Biomedical Sciences, University of Bern, Bern, SWITZERLAND;3 Institute for Virology and Immunobiology, University of Würzburg, Würzburg,GERMANYThe measles virus (MeV) and canine distemper virus (CDV) entry systemsare regulated by two envelope glycoproteins: the attachment (H)and fusion (F) proteins, which tightly co operate to mediate membranefusion. While MeV is still associated with over 120 ′ 000 human deathsper year, CDV has an ever increasing host range with recently reportedsevere outbreaks in primates. Inefficient vaccine availability in developingcountries combined with the overarching increasing tendency of relaxedmeasles vaccination in developed countries may set the stage for swiftadaptation of CDV to humans with putative dramatic global health outcomes.We here refined the molecular basis of CDV adaptation to thehuman SLAM (hSLAM) receptor. Indeed, we recently reported that, initially,CDV poorly infects hSLAM expressing cells with the requirementof a single substitution in CDV H to enable efficient CDV infections.Here, we found that wt CDV H nevertheless engaged to hSLAM. However,the avidity of the CDV H/hSLAM interaction was rather low, whichprecluded productive membrane fusion triggering. We identified threeresidues in hSLAM locating at the binding interface which, when mutatedinto the canine SLAM corresponding amino acid (E71G, E75K andR130H), allowed wt CDV glycoproteins to promote fusion without improvingthe weak CDV H/receptor interaction. Remarkably, fusion was alsoachieved when the CDV H/hSLAM combination was coupled with destabilized CDV F variants. Overall, our data revealed that only minormodifications in hSLAM, CDV H or CDV F lead to efficient cell entrytrough the human receptor. The ease of CDV adaptation to heterotypicreceptors may hence contribute to its threatening, ever increasing, hostrange.REF 157Impact of nectin 1 during entry of Herpes simplex virus type 1 intoepidermis and keratinocytesDagmar KNEBEL MÖRSDORF 1,2 , Philipp PETERMANN 1 , KatharinaTHIER 1 , Elena RAHN 1 , Wilhelm BLOCH 3 , Martin J. BARRON 4 ,Michael J. DIXON 41 Center for Biochemistry, University of Cologne, Cologne, GER-MANY; 2 Department of Dermatology, University of Cologne, Cologne,GERMANY; 3 Department of Molecular and Cellular Sports Medicine,German Sports University, Cologne, GERMANY; 4 Faculty of LifeSciences, University of Manchester, Manchester, UNITED KINGDOMKeratinocytes of the skin and mucosa represent the primary entry sitefor herpes simplex virus type 1 (HSV 1) in vivo. We aim to identify themolecular mechanisms on which virus entry depends in keratinocytes tounderstand how HSV 1 overcomes the barrier function of the epithelia.Here, we used a mouse model to address the role of nectin 1, one ofthe major co receptors for HSV 1 in various cell lines. Virus entry wasstudied after ex vivo infection of murine epidermal sheets by visualizingexpression of the immediate early gene ICP0. The studies are based onour observation that infection initiates efficiently in the basal keratinocytesof the epidermis once the dermis is removed. EM studies suggestvirus internalization via fusion of the viral envelope with the basal surfaceof the epidermal sheets and endocytic uptake. Our results in nectin 1deficient epidermal sheets revealed a 70 90% reduction in the number ofinfected keratinocytes in comparison with wildtype epidermis. Interestingly,we observed nearly no infection in isolated nectin 1 deficient primarykeratinocytes. This observation correlated with the presence of HVEM, afurther HSV 1 co receptor, in murine epidermis and the lack of HVEMexpression in primary murine keratinocytes. Overexpression of HVEM innectin 1 deficient keratinocytes restored infectivity. Thus, we suggest thatHVEM supports entry into a subpopulation of cells in nectin 1 deficientepidermis while the absence of nectin 1 and HVEM in primary keratinocytesleads to a loss of entry. In summary, our results demonstrate a majorimpact of nectin 1 as co receptor for HSV 1 in murine epidermis and asupplementary role of HVEM.S162 Virologie, Vol 17, supplément 2, septembre 2013