rologie i - European Congress of Virology

5 th European Congress of VirologyThursday 12 th September 2013,15h00 – 17h15WORKSHOP 14: “VIRUS STRUCTURE, DYNAMICIMAGING AND TRAFFICKING”KEYNOTE:Chairpersons: Paul DUPREX (Boston, USA)& François PENIN (Lyon, FRANCE)Room Tête d’OrParamyxoviral ultrastructures revealed by electron cryotomographyLassi LILJEROOS 1 , Juha T. HUISKONEN 2 , Magdalena A.KRZYZANIAK 4 , Ari ORA 1 , Petri SUSI 3 , Ari HELENIUS 4 and SarahJ. BUTCHER 11 Institute of Biotechnology, University of Helsinki, 00790, Helsinki, FIN-LAND; 2 Oxford Particle Imaging Centre, Division of Structural Biology,Wellcome Trust Centre for Human Genetics, University of Oxford, OxfordOX3 7BN, UNITED KINGDOM; 3 Department of Virology, University ofTurku, 20520, Turku, Finland; 4 Institute of Biochemistry, ETH Zurich,CH-8092 Zurich, SWITZERLANDParamyxoviruses include several significant human pathogens, likemeasles, mumps and respiratory syncytial viruses. Due to the highly pleomorphicnature of these viruses, three-dimensional whole-virus structureshave been missing until recently. Electron cryotomography is a suitablemethod to undertake such structural studies and reveal details of the componentsconstructing the virion. We have applied electron cryotomographytogether with subtomographic alignment and averaging to characterize theultrastructures of measles and respiratory syncytial viruses. We show thatthese two related viruses have significant differences in the way the matrixprotein is organized within the virion. We also discuss the implications ofthese differences on virion assembly.ORAL COMMUNICATIONSREF O56Real time cell biological analysis of vaccinia replicationN. Bishara MARZOOK, Timothy NEWSOME, ChristopherMCKENZIE, Helena LYNN, Dean PROCTER, JacquelynHORSINGTONSchool of Molecular Bioscience/University of Sydney, Sydney, AUSTRA-LIAThe fluorescent labeling of viruses is an important tool in the study of hostpathogen relationships. Vaccinia virus, the live vaccine used in the eradicationof smallpox, has proven particularly amenable to live cell microscopyowing to the ease with which it able to be engineered at the genome level.We report here a novel method that greatly reduces the number of stepsrequired in the process of creating fluorescent recombinant viruses. Thistechnique combines the ability of successful homologous recombinationoccurring using relatively short regions of homology with the principle oftransient dominant selection (TDS) employing selectable metabolic andfluorescent markers. We believe this technique provides a relatively rapid,robust and reliable method of fluorescently labeling viral proteins of interestor different cellular structures in a modular manner. Double or triplelabeled recombinant viruses can also be generated by co infection andpurification based on fluorescence. We will present our latest fluorescentviruses that can be used to visualise a range of host and viral structuresduring virus replication.REF O57Intracytoplasmic transport of hepatitis B virus capsids and their dissociationfrom microtubuleQuentin OSSEMAN 1 , Birgit RABE 2 , Aurelia CASSANY 1 , HaraldWODRICH 1 , Elodie BERDANCE 1 , Dominique BEGU 1 , IrinaSOWINSKEYA 3 , Paul PUMPENS 3 , Andris DISEHLER 3 , MichaelKANN 11 institute of Fundamental Microbiology and Pathgenicity (CNRS UMR5234), Bordeaux, FRANCE; 2 Institute of Medical Virology, Giessen, GER-MANY; 3 Latvian Biomedical Research and Study Centre, Riga, LATVIAHepatitis B virus (HBV) is a pararetrovirus which needs the transcriptionmachinery of the cell nucleus for replication. The virus thus depends on thetransport its genome from the cell periphery to the nuclear envelope. Theretrograde intracytoplasmic trafficking is facilitated along microtubules(MT) using motor protein complexes of the dynein family, which composesof 14 protein chains.We showed by lipofection of capsids that they transport the HBV genometo the nuclear pore leading to release of the genome exclusively into thenucleus. Intracytoplasmic transport was dependent upon intact MT.Using the different dynein light and intermediate chains expressed inE. coli we identified one light chain (DYNLL1, member of the LC8 family)specifically interacting with mature capsids only. DYNLL1 differs fromDYNLL2, which did not interact with the capsids, by six amino acids only.Single directed mutagenesis on each amino acid show that in fact L29 andH41 were important for direct interaction between capsids and DYNLL1.DYNLL1, also called hub protein, is known to interact with 66 cellularpartners via a common short linear motif which also interacts with thedynein intermediate chains (DYNIC). The binding of these interactionpartners is thus incompatible with a function in intracytoplasmic transportas their binding is competitive to dynein binding. We thus propose thatHBV capsid binding to DYNLL1 occurs in addition to DYNLL1 DYNDICinteraction, which would be the first example of a cargo attachment todynein via DYNLL1.REF O58Tracking genomes of DNA viruses in the cytosol and nucleus by clickchemistry and super resolution microscopyI. Hsuan WANG 1 , Maarit SUOMALAINEN 1 , Samuel KILCHER 2 ,Jason MERCER 2 , Anne NEEF 3 , Nathan LUEDTKE 3 , Urs F. GREBER 11 Institute of Molecular Life Sciences, University of Zurich, Zurich,SWITZERLAND; 2 Institute of Biochemistry, ETH Zurich, Zurich, SWIT-ZERLAND; 3 Institute of Organic Chemistry, University of Zurich, Zurich,SWITZERLANDHow viral genomes access the cytosol or nucleus, or are destroyed ispoorly known, largely because they have been difficult to track. Herewe used metabolic labelling and click chemistry to track the genomesof three families of DNA viruses, adenovirus, vaccinia virus and herpesvirus. The nucleosides 7 deaza 7 ethynyl 2 ′ deoxyadenosine (EdA) and2 ′ deoxy 5 ethynylcytidine (EdC) incorporated into adenovirus and herpesvirusreplication sites, as demonstrated by copper(I) catalyzed azide –alkyne (click) reactions with fluorescent azides, and gave normal adenovirusyields, unlike 5 ethynyl 2’ deoxyuridine (EdU). EdA and EdC alsointegrated into adenovirus particles. EdU but not (2 ′ 5) 2 ′ deoxy 2 ′ fluoro5 ethynyluridine (F ara EdU) was readily incorporated into vaccinia virusreplication centers and virions. In extracellular adenovirus, ethynyl taggedDNA was fully protected, but accessible to azide fluorophores in cytosolicviruses, as shown by g STED super resolution microscopy. Aftervirus disassembly at the nuclear pore complex, capsid free viral DNAwas found in the nucleus, but also in the cytosol suggesting that nuclearimport of viral DNA is a bottleneck. Our results show that click chemistrycompatible nucleoside analogues give fully infectious virus particles, andsuperior resolution and sensitivity in detecting single viral DNA genomesS62 Virologie, Vol 17, supplément 2, septembre 2013