rologie i - European Congress of Virology

5 th European Congress of Virology25. RESPIRATORY VIRUS INFECTIONSPosters: REF 508 to REF 538REF 508Candidate plant derived universal Influenza A nanovaccines conferprotection against lethal homologous and heterologous virus challengePeter IVANOV, Tatiana GASANOVA, Natalia PETUKHOVALomonosov Moscow State University, Moscow, RUSSIAConservative Influenza M2e antigen (23 aa) has been overexpressed onthe surface of tobacco mosaic virus (TMV U1) particles. Three versionsof M2e were inserted into the TMV coat protein (CP) gene. In additionto the consensus human M2e sequence, we introduced mutations by thesubstitution of cysteines with either serine or alanine residues. Agrobacteriummediated infections of Nicotiana benthamiana produced as much as4 g of TMV M2e ala per 1 kg of systemic leaves. Genetic stability of viralvectors was demonstrated. Chimeric virions consisted of two proteins andcontained up to 90% of CP M2e. Immunized mice did not lose weightcomparing with the control animals. The amount of antibodies specificto M2e exceeded those specific to TMV particle itself (maximum ratio5/1). Immunogold microscopy proved that the foreign antigens were uniformlydistributed and tightly packed on the surface of chimeric particles.The majority of TMV CP epitopes are hidden from the immune systemby the M2e antigens exposed on the particle surface. IgG1/IgG2a ratiovaries from 0.7 (Ser) to 3.2 (Ala). The anti M2e IgG response declinedinsignificantly during the 7 month period. Vaccinated mice were resistantto 5 LD50 of A/PR/8/34 (H1N1) and TMV M2e ala conferred partial protection(70% of survival rate) against heterologous strain (4 aa changes inM2e) influenza A/California/07/2009 (H1N1). Virus titer in lungs was twoorders lower than in the non immune animals. Therefore, a new generationcandidate universal Influenza A nanovaccines with specific rod shapedepitope geometry has been obtained.REF 509R.I.P. Rest in Peace: Human rhinoviruses target the death kinaseRIPK1 to interfere with caspase dependent cell deathMark LÖTZERICH, Urs F. GREBERInstitute of Molecular Life Sciences, University of Zurich, Zurich, SWIT-ZERLANDVirus infections can trigger apoptosis by activation of death receptorsor pattern recognition receptors, such as tumor necrosis factor receptor(TNFR), double strand RNA sensor toll like receptor 3 (TLR3) or RNAhelicase melanoma differentiation associated gene 5 (MDA5). Besides initiatorcaspase 8 and executor caspases this can involve regulatory kinases,such as receptor interacting protein kinase 1 (RIPK1). RIPK1 transmitsapoptotic signals via its death domain to caspase 8. We addressed howhuman rhinoviruses (HRV1a, 2, 14, 16, 37) interfere with cell death processesin Hela OHIO and primary human nasal epithelial cells. HRVinfection induced low levels of activation of caspases 8 and 9. Host chromatinrelocalized to the nuclear periphery and lamins were partially cleavedindependent of caspase activity. There was no sign of DNA fragmentation,poly ADP ribose polymerase cleavage, or activation cleavage of caspases3 and 7. Instead, the death domain of RIPK1 was cleaved to 60, 47 and37 kDa fragments. Ruptintrivir, an inhibitor of HRV 3C protease blockedthe formation of the 60 kDa RIPK1 fragment and abrogated cytopathiceffects. RIPK1 was found in a complex with 3C protease in infected cellsand recombinant 3C protease cleaved RIPK1. Collectively, the data suggestthat HRV induces upstream activation signals for apoptosis but blockexecution of apoptosis by 3C protease mediated cleavage of RIPK1. Thisleads to a non classical death pathway, which is controlled by viral genesrather than the host apoptosis machinery.REF 510Analyzing the receptor binding of influenza A viruses by using solublechimeric proteinsAnne Kathrin SAUER 1 , Chi Hui LIANG 2 , Maren BOHM 1 , ChristelSCHWEGMANN WESSELS 1 , Chung Yi WU 2 , Chi Huey WONG 2 ,Georg HERRLER 11 Institute of Virology, University of Veterinary Medicine, Hannover, GER-MANY; 2 Genomics Research Center, Academia Sinica, Taipei, TAIWANInfluenza A viruses can be found in many avian and mammalian speciesand are known for their ability to cross species barriers and adapt to newhosts. One major aspect for switching the host is the binding specificityof the surface glycoprotein hemagglutinin. It binds to cell surface sialicacids and viruses from different host species preferentially recognize sialicacids in certain linkage types. Avian influenza strains generally showa preference for 2,3 linked sialic acids whereas human strains show ahigher affinity to sialic acids linked in 2,6 conformation to galactose.Many studies use two plant lectins to stain for 2,3 and 2,6 linked sialicacids in cells and tissues: SNA (Sambucus nigra agglutinin) and MAA(Maackia amurensis agglutinin). But these two lectins have of course theirown individual binding properties. Using only these lectins is not sufficientto analyze the binding properties of HAs considering the high amount ofdifferent oligosaccharides even on a single cell and the variety of differentHA subtypes. To circumvent this problem we utilize soluble forms of severalHA subtypes to investigate the binding properties of the influenza HA,exploiting the receptor specificity of each subtype. Soluble hemagglutininsof the avian subtypes H5, H7 and H9 as well as human and porcineH1 subtypes have been tested on different cell lines and tissue sectionsfrom avian trachea and the porcine respiratory tract. Glycan array analysisof solHAs and lectins was performed to get a detailed insight into receptorbinding properties of influenza HAs.REF 511Efficient uncoating of influenza A virus requires exposure to mildlyacidic pH and a high concentration of potassium prior to fusion inlate endosomesSarah STAUFFER 1 , Yuehan FENG 1 , Firat NEBIOGLU 1 , LassiLILJEROOS 2 , Butcher SARAH J. 2 , Paola PICOTTI 1 , Ari HELENIUS 11 Institute of Biochemistry, ETH Zurich, Zurich, SWITZERLAND;2 Institute of Biotechnology, University of Helsinki, Helsinki, FINLANDFollowing endocytic uptake, Influenza A virus (IAV) is transported tolate endosomes where virus fusion is induced by the viral hemagglutinin,with an optimum of pH 5.0 for the strain X31 (A/Aichi/2/1968 (H3N2)).Acidification of the viral core in endosomes prior to fusion promotes efficientviral ribonucleoprotein (vRNP) uncoating. At mildly acidic pH theM2 channel mediates proton translocation into the viral core, resulting indisassembly of matrix protein 1 (M1) oligomers and release of vRNPsinto the cytoplasm following fusion. To date, core specific viral proteinprotein interactions and their dissociation upon acid exposure have mainlybeen analyzed in vitro. Here, we aimed to assess the pre fusion requirementsof IAV uncoating in the context of infection, using pH 5.0 mediatedfusion at the plasma membrane. Mild acidification (pH 5.8) as well astreatment of IAV at high K+ concentrations mimicking the endosomalenvironment prior to fusion increased viral uncoating and infection significantly.In particular, core disassembly in vitro and in vivo, as monitoredby M1 dissociation and vRNP exposure, was facilitated when virions werepre acidified. We applied limited proteolysis combined with targeted massspectrometry in order to probe conformational changes in the virion. M1S262 Virologie, Vol 17, supplément 2, septembre 2013