rologie i - European Congress of Virology

5 th European Congress of Virologylambda2 in the inhibition of replication of WNV (lineages 1 and 2) andin the activation of intracellular pathways of IFN response in differentcell lines. To this aim, A549 and Vero E6 cells were treated with increasingamounts of IFN lambda either IFN lambda1a or lambda2a or IFNalpha, used alone or in combination, then infected with WNV 1 or 2; theextent of virus yield inhibition was established; mRNA levels of MxA and2 ′ 5 ′ OAS were measured as well. The antiviral potency of IFN lambda1and lambda2 was lower than that of IFN alpha. When IFN alpha andlambda were used together, the Combination Index (CI) for virus inhibitionwas >1 (although more pronounced in Vero E6 cells infected with WNVlineage 2), indicating antagonistic effect. The same results were obtainedusing other viruses (Chikungunya virus, EMCV and HSV 1). Antagonismbetween IFN alpha and lambda was also observed for the inductionof mRNA for both MxA and 2 ′ 5 ′ OAS. Elucidating the interplay betweenIFN alpha and lambda may help to better understand innate defencemechanisms against viral infections, including the molecular mechanismsunderlying the possible influence of IL-28B polymorphisms on the naturalhistory of flavivirus infections. This work was partially supported by theEuropean Union Seventh Framework Programme [FP7/2007-2013] underGrant Agreement n ◦ 278433-PREDEMICS.REF 020The measles virus phosphoprotein interacts with the linker domainof STAT1Patricia DEVAUX, Lauren PRINISKI, Roberto CATTANEOMayo Clinic, Rochester, MN, USAThe measles virus (MV) phosphoprotein (P) and V proteins block the interferon(IFN) response by impeding phosphorylation of the signal transducerand activator of transcription 1 (STAT1) by the Janus kinases (JAKs). Wesought to characterize here how STAT1 interacts with MV P. Based onsequence and function conservation between MV and Nipah virus (NiV)P proteins, we postulated that the region of STAT1 interacting with NiV P(residues 509 to 712) also interacts with MV P. Within this region, sinceMV P does not interact with STAT2, we mutated segments that have nohomology to it. These segments cover the linker domain, the Src homology2 (SH2) domain and the transactivation domain. We initially producedmutants of two or three residues within these segments, and then alsomutants of individual residues. The expression and effect of these mutantswere tested in STAT1 deficient cells. Certain mutants of the linker, theSrc homology 2 domain (SH2), or the transactivation domain had reducedor abolished phosphorylation through JAKs after IFN treatment. Othermutants, mainly localized in the linker, failed to interact with P as documentedby the lack of interference with nuclear translocation. Thus, thefunctional footprint of P on STAT1 localizes mainly to the linker domain;there is also some overlap with the STAT1 phosphorylation functionalfootprint on the SH2 domain. Based on these observations, we propose amodel of how the MV P and V proteins may operate in concert to inhibitthe JAK/STAT pathway.REF 021Mechanism of inhibition of the innate immune response by alphavirusnsP2 proteinElena FROLOVA 1 , Ivan AKHRYMUK 1 , Sergey KULEMZIN 11 University of Alabama at Birmingham, Birmingham, USAOutcome of viral infection at the cellular level depends on competitionbetween cellular antiviral response and ability of the virus to inhibit itsactivation. The antiviral response induces pathways mediating non cytopathicclearance of the virus from infected cells and release of interferon andother cytokines, which establish the antiviral state in yet uninfected cells.Numerous viruses promote infection spread and viremia development by atleast partially inhibiting cellular antiviral response. It has been previouslydemonstrated that the Old World alphaviruses, including Sindbis, SemlikiForest and chikungunya viruses, evade the innate immune responseby inducing rapid and global inhibition of cellular transcription, whichprevents activation of cellular antiviral genes. We have defined the mechanismof transcriptional shutoff utilized by the Old World alphaviruses.During virus replication, the nonstructural protein nsP2 is transported intothe nucleus of the infected cells, where it induces rapid degradation ofthe catalytic subunit of the DNA dependent RNA polymerase Rpb1. Wefound that degradation of Rpb1 does not depend on the nsP2 associatedprotease activity. Instead, nsP2 imitates activation of the transcriptioncoupled repair mechanism, which leads to complete, ubiquitin dependentdegradation of Rpb1 within 4 6 h post infection. Interestingly, the NTPaseactivity of nsP2 helicase domain is critical for Rpb1 degradation. Thus,the Old World alphaviruses employ a previously unknown and uniquemechanism for robust inhibition of cellular antiviral response.REF 022Characterization of the effect of antiviral enzyme RNase L on differentpathogenic strains of influenza A virusWai Yip LAM 1 , Denis Pc CHAN 2 , Paul Ks CHAN 2,3 , Stephen KwTSUI 11 School of Biomedical Sciences, Faculty of Medicine, The Chinese Universityof Hong Kong, Shatin, HONG KONG; 2 Stanley Ho Centre forEmerging Infectious Diseases, Faculty of Medicine, The Chinese Universityof Hong Kong, Shatin, HONG KONG; 3 Department of Microbiology,Faculty of Medicine, The Chinese University of Hong Kong, Shatin, HONGKONGInfluenza A viruses cause a highly contagious respiratory disease inhumans and are responsible for the periodic widespread pandemics, thathave caused high mortality rates. Recently, the World Health Organization(WHO) has recorded a mortality rate of greater than 60% in thehighly pathogenic avian influenza A (H5N1) virus infection. The fatal outcomeof human influenza A H5N1 is found to be associated with reactivehaemophagocytic syndrome and multi organ failure mediated by hypercytokinemia.Ribonuclease L (RNase L) is a latent endoribonuclease in theinterferon regulated double strand RNA (dsRNA) activated antiviral 2 ′ 5 ′oligoadenylate synthetase (OAS)/RNase L pathway. Activated RNase Lwill cleave viral single strand RNA (ssRNA) predominantly at single strandedUA and UU dinucleotides position(s), resulting in small, often duplexRNA cleavage products. These small RNA cleavage products can furtheractivate the interferon (IFN) as well as other cytokines responses via specificpathogen recognition receptors, such as retinoic acid inducible gene I(RIG I), melanoma differentiation associated gene 5 (MDA5) and toll likereceptors (TLR). We postulate that different viral RNA sequences fromdifferent pathogenic strains of influenza A virus will be cleaved by RNaseL in a sequence specific manner and the resulting amount of cleavage RNAproducts will thus be different. Bioinformatic data demonstrating a quantitativedifference between the number of UA/UU sites between highlypathogenic and low pathogenic viruses. The results were further verifiedusing an in vitro model.REF 023Mononegavirales leader RNA as agonist of RIG IJade LOUBER 1 , Louis Marie BLOYET 1 , Eva KOWALINSKI 2,3 ,Stephen CUSACK 2,3 , Denis GERLIER 11 Centre International de Recherche en Infectiologie, CIRI, INSERMU111, CNRS UMR5308, Université Lyon 1, ENS Lyon, Lyon, FRANCE;2 European Laboratory of Molecular Biology, Grenoble, FRANCE; 3 VirusCell host Interactions Unit, UJF EMBL CNRS, UMI 3265, Grenoble,FRANCES124 Virologie, Vol 17, supplément 2, septembre 2013