rologie i - European Congress of Virology

5 th European Congress of VirologyThe tyrosine based sorting motif in the cytoplasmic tail of the TGEV Sprotein is important for its retention in the ERGIC, the site of coronavirusbudding. This tyrosine based motif is well conserved in S proteins ofalphacoronaviruses. To study the role of this sorting motif (1440YXXI) ininfectivity, in incorporation of S protein into virus particles and in interactionwith the coronavirus M protein, the motif was disrupted with a tyrosineto alanine mutation (Y/A). A lysine to methionine mutation (K/M) at position1444 transformed the tyrosine based retention signal into a tyrosinebased endocytosis signal.Recombinant TGEV were constructed and analyzed for infectivity. Thevirus with a disrupted tyrosine motif (YI/AA) in the S protein was not ableto infect ST cells whereas the wildtype virus did. A complementation ofthe non infectious rTGEV with authentic S protein or the K/M mutant ledto the production of infectious virus. We analyzed whether the tyrosinebased motif is important for interaction with the M protein. The authenticS as well as as both mutants (Y/A, K/M) could interact with M as shown byimmunofluorescence and western blot analysis. In a VLP assay we coulddemonstrate that the Y/A and YI/AA mutant was not incorporated intoparticles while authentic S and K/M could be found together with M inVLPs.We conclude that the tyrosine based sorting signal in the TGEV S proteinis essential for incorporation of S into virions. The interaction with so farunknown cellular proteins via this motif seems to be one key factor for theassembly of infectious virus.REF O139Essential role of the Fusion protein during Sendai virus particle formationManel ESSAIDI LAZIOSI 1 , Anastasia SHEVTSOVA 1 , DenisGERLIER 2 , Laurent ROUX 11 Department of Microbiology and Molecular Medicine, Faculty of Medicine,University of Geneva, Geneva, SWITZERLAND; 2 Ecole NormaleSupérieure de Lyon, INSERM U1111, CNRS UMR 5308, Lyon, FRANCEPrevious studies have shown the importance of the fusion protein (F)during Sendai virus particle production. The absence of the TYTLE motifin the cytoplasmic domain of F has the same deleterious effect on VPproduction as the suppression of the entire F protein (Fouillot Coriou, N.and Roux, L. 2000). The mechanism by which this motif is involved inthis process is not understood. We demonstrate by confocal microscopyand immunoprecipitation that a F protein harboring 5 alanines instead ofthe TYTLE motif is not express at the cell surface although it is still ableto interact with the matrix protein (M). Interestingly, the absence of themutated F at the cell surface correlates with a lower representation of HNand M at cell surface. In addition, these two proteins show a more diffusedand disorganized pattern, both at the cell surface and in the cytoplasm. Inthe end, the mutated F retains M in the cytoplasm and prevents the processof assembly. These results highlight the important role of the fusionprotein during virus particle formation.REF O138Association of apolipoprotein B with Hepatitis C virus is involvedin viral infectivity and is dependent on the liver enriched proteinCidebChristophe RAMIÈRE 1,2,3 , Liviu Sorin ENACHE 4 , MarinePORCHEROT 2,3 , Olivier DIAZ 2,3 , Laure PERRIN COCON 2,3 , VincaICARD 1 , Caroline SCHOLTÈS 1,2,3 , Vincent LOTTEAU 2,3 , PatriceANDRÉ 1,2,31 Hospices Civils de Lyon, Lyon, FRANCE; 2 INSERM U1111, CIRI, Lyon,FRANCE; 3 Université de Lyon, Lyon, FRANCE; 4 University of Medicineand Pharmacy, TârguMure, ROMANIAIn patients’ blood, Hepatitis C Virus (HCV) circulates as lipo viral particles(LVP), hybrid particles associating viral and lipoprotein components, inparticular apolipoprotein B (apoB). Contrary to natural LVPs, HCV particlesproduced in vitro in Huh7.5 cells (HCVcc) are characterized bypoor association with apoB and low specific infectivity. We thus hypothesizedthat apoB could be involved in HCV infectivity and examinedthe mechanisms responsible for its association with HCV virions. We firstdemonstrated by neutralization experiments that 2 monoclonal anti apoBantibodies, recognizing epitopes in the Low–Density Lipoproteins Receptorbinding domain of apoB, were able to strongly neutralize (ca 80 to90%) infection of Huh7.5 cells by HCVcc, contrary to antibodies targetingepitopes outside this domain. We also identified Cideb, a liver enrichedprotein involved in lipoprotein assembly, as an essential factor for associationof HCV with apoB. We showed that Cideb directly interacted withHCV E2 envelope glycoprotein. We also observed that Cideb expressionwas low in Huh7.5 cells and that Cideb overexpression in this cell line wassufficient to induce secretion of E2 in association with apoB. Moreover,differentiation of Huh7.5 cells in presence of human serum and DMSO ledto a dramatic increase of Cideb expression and further secretion of HCVparticles characterized by higher association with apoB and improved specificinfectivity. Together these results suggest that apoB plays an importantrole in HCV entry and that Cideb is essential for HCV association withapoB.REF O140A comparative functional RNA interference screen identifies factorsdifferentially required for lipid droplet homeostasis and life cycle ofFlaviviridae membersGualtiero ALVISI 1,2 , Ina Karen STOECK 2 , Sandeep AMBERKAR 3 ,Narsis Aftab KIANI 3 , Christoph SOMMER 4 , Wolfgang FISCHL 2 ,Marion POENISCH 2 , Fred HAMPRECHT 4 , Giorgio PALÙ 1 , LarsKADERALI 4 , Ralf BARTENSCHLAGER 21 Department of Molecular Medicine, University of Padua, Padua, ITALY;2 Department of Molecular Virology, University of Heidelberg, Heidelberg,GERMANY; 3 Institute of Medical Informatics & Biometry, Dresden Universityof Technology, Dresden, GERMANY; 4 Heidelberg Collaboratoryfor Image Processing, University of Heidelberg, Heidelberg, GERMANYLipid Droplets (LDs) play a central role in storage and mobilization oflipids and are involved in lipid metabolism related diseases, as well as inthe replication cycle of several viruses, including the hepatotropic hepatitisC virus (HCV) and the Dengue virus (DENV). However, in spiteof their importance, the pathways and factors regulating LD homeostasisin human liver cells are poorly characterized, and their suitability asantiviral targets has not been explored. To overcome this limitation, weassembled a siRNA library targeting 230 genes potentially regulating LDhomeostasis. This library was used to perform a comparative functionalRNA interference screen to identify key factors of LD homeostasis waswell as factors promoting or restricting HCV and DENV replication. Wecould identify 59 genes as important factors for LD homeostasis, most ofthem also playing key roles for viral replication. Bioinformatic analysisof hits identified biological processes regulating both viral life cycle andLD homeostasis. These include COPI coated vesicle budding, RNA splicingor proteasomal and ubiquitin dependent catabolic processes. Uponconfirmation by a secondary deconvolution screen, the DDX3 dead boxhelicase and the calcium independent phospholipase A2 iPLA2 wereselected for follow up studies. Our results suggest a novel role for DDX3in HCV assembly/release, which appears to be independent from its interactionwith core protein. Moreover, pharmacological ablation of iPLAactivity selectively impaired HCV particle production, but did not affectthe DENV life cycleS98 Virologie, Vol 17, supplément 2, septembre 2013