rologie i - European Congress of Virology

5 th European Congress of Virology14. VIRUS STRUCTURE, DYNAMICIMAGING AND TRAFFICKINGPosters: REF 216 to REF 229REF 216Structural and functional studies of the N terminal dimerizationdomain of the Epstein Barr virus nuclear antigen 2Sybille THUMANN 1 , Anders FRIBERG 2 , Sybille THUMANN 1 , PeijianZOU 2 , Janosch HENNIG 2 , Michael SATTLER 2 , Bettina KEMPKES 11 Department of Gene Vectors, Helmholtz Center Munich, GermanResearch Center for Environmental Health, Munich, GERMANY;2 Institute of Structural Biology, Helmholtz Center Munich, GermanResearch Center for Environmental Health, Munich, GERMANYEpstein Barr virus (EBV) is a gammaherpesvirus that establishes lifelongasymptomatic infections in the majority of human adults. It is the causativeagent of infectious mononucleosis if primary infection is delayed toadolescence. Furthermore, epidemiological and molecular evidence linksEBV to the pathogenesis of endemic Burkitt ′ s lymphoma, nasopharyngealcarcinoma, a subset of Hodgkin ′ s disease, and other malignanciesof lymphoid and epithelial cell origin. EBV has the unique ability totransform resting B cells into permanently proliferating, latently infectedlymphoblastoid cell lines that express six EBV nuclear antigens and3 latent membrane proteins. One of the first nuclear antigens expressedpost infection is the Epstein Barr virus nuclear antigen 2 (EBNA2). It canform dimers and transactivates a set of viral and cellular genes. Here weanalyzed the EBNA2 N terminal dimerization domain (END). NMR spectraof the END domain were recorded and revealed that the dimerizationof the monomers is presumably driven by the formation of a hydrophobiccore. Based on this structure, interface and surface mutants of theEND domain were generated. They were tested for their capacity to selfassociate by co immunoprecipitation and for their ability to transactivateEBNA2 target genes in a Burkitt ′ s lymphoma cell line. Interestingly, notonly interface mutations that impair dimerization, but also surface mutationsof the END domain prevent biological activity of the transactivatorEBNA2.REF 217Structural virology of the murid Gamma Herpesvirus 4Bruno CORREIA 1 , Colin MCVEY 1 , Marta MIRANDA 21 Institute of Chemical and Biological Technology, Lisbon, PORTUGAL;2 Institute of Molecular Medicine, Lisbon, PORTUGALThe murid herpesvirus 4 (MuHV4) has been widely explored as a modelfor research on the human gamma herpesvirus pathogenesis due to itsability to infect mice via the nasal passages and also because it is geneticallyrelated to other malignancy associated pathogens such as the humanEpstain Barr virus (EBV) and Kaposi’s Sarcoma herpesvirus (KSHV).As for EBV, MuHV4 establishes a lifelong latency stage in the nucleusof host B cells as a multicopy episome and only a small fraction ofviral genes are expressed. Amongst this fraction of genes, an ORF protein,ORF73, was demonstrated to promote a deficit on the establishmentof latency in vivo when mutant viruses failed to express them. ORF73reveals remarkable sequence homology with KSHV latency associatednuclear antigen (LANA) and striking predicted structural homology withthe C terminal domain of the EBV nuclear antigen 1 (EBNA1), mutuallycrucial for viral latency by tethering the viral episome to host chromosomes,therefore ensuring replication and segregation of the viralgenome to daughter cells. C terminal LANA and EBNA1 were describedto bind terminal repeat (TR) DNA of the viral genome upon dimerization,suggesting that C terminal of ORF73 may also be involved inepisome maintenance functions. We crystallized and solved the first X raycrystallographic structure of the murid ORF73 protein unbound to viralDNA and highlighted important residues and structural motifs that mayexert functional relevance as therapeutic targets by manipulation of virallatency.REF 218Visualization of the pre integration complex of the murine leukemiavirus during early steps of infectionEran BACHARACH 1 , Efrat ELIS 1 , Marcelo EHRLICH 1 , Darren J.WIGHT 2 , Virginie C. BOUCHERIT 2 , Adi PRIZAN RAVID 1 , NihayLAHAM KARAM 1 , Kate N. BISHOP 21 Tel Aviv University, Tel Aviv, ISRAEL; 2 MRC National Institute for MedicalResearch, London, UNITED KINGDOMTo integrate, reverse transcribed retroviral genomes are imported fromthe cytoplasm to the chromosomes as part of a pre integration complex(PIC). Differences in retrovirus PIC trafficking influence their ability toinfect resting and/or dividing cells. Lentiviruses, including HIV, infectboth dividing and resting cells. In contrast, simple oncoretroviruses, suchas murine leukemia viruses (MLVs), are restricted to dividing cells. p12,a cleavage product of MLV Gag precursor, is thought to influence MLVintegration. Viruses with specific lethal mutations in p12 showed defectsin early stages of infection, with normal generation of linear genomicDNA, but no formation of circular DNA forms (which are thought to marknuclear entry of the viral DNA). In the past we demonstrated that p12is a functional component of the MLV PIC and recently we also revealeda role for p12 as a tethering factor, which connects this PIC to mitoticchromosomes. The fact that p12 escorts the MLV DNA throughoutthe early stages of infection promoted us to microscopically detect theincoming MLV PIC by labeling p12. This allowed us the visualization ofthe PIC both by immunofluorescence and by real time imaging. Here wedescribe the possible connection of PIC movements to the cytoskeleton,PIC trafficking in respect to changes in the cell cycle, the possible contributionof p12 to PIC stability and the gradual uncoating of the PIC duringits trafficking. Altogether these results highlight steps important for MLVintegration, which also contribute to the high tropism of MLV to dividingcells.REF 219Structural characterization of the Influenza virus matrix protein M1Jens RADZIMANOWSKI 1 , Marta BALLY 2 , Eric CONDAMINE 3 ,Stéphanie MANGENOT 2 , Patricia BASSEREAU 2 , WinfriedWEISSENHORN 11 Unit of Virus Host Cell Interactions (UVHCI), Grenoble, FRANCE;2 Institut Curie, Paris, FRANCE; 3 Institut de Biologie Structurale (IBS),Grenoble, FRANCEInfluenza viruses (genus A, B and C) are negative strand segmentedRNA viruses, which acquire their envelope from the plasma membrane.Although the matrix protein M1 is an integral part of infectious maturevirions and forms a helical protein layer underneath the viral membrane,its role in assembly and budding is poorly understood. Unlike othermatrix proteins from enveloped viruses M1 expression alone does notinduce VLP formation. We present structural data on M1 and in vitromembrane binding to better understand the role of M1 in the viral lifeS180 Virologie, Vol 17, supplément 2, septembre 2013