ID: 106

Abstract / Cytokine 76 (2015) 66–112 receptor is the RNA helicase RIG-I, which has the ability to detect and be activated by 5’triphosphate uncapped double stranded RNA (dsRNA) as well as the viral mimic dsRNA polyI:C. Once activated, RIG-I’s CARD domains oligomerize and initiate downstream MAVS signaling ultimately inducing interferon (IFN) production. Another dsRNA binding protein PACT, originally identified as the cellular protein activator of PKR, has recently been shown to stimulate RIG-I signaling in response to polyI:C treatment, in part by stimulating RIG-I’s ATPase and helicase activities and resulting in an enhanced induction of IFN. TRBP (TAR-RNA-binding protein), which is about 45% homologous to PACT is known to inhibit PKR signaling by sequestration of PKR and its’ activators, dsRNA and PACT. Despite the domain homology and similar structure of PACT and TRBP, the role of TRBP is yet to be explored in RIG-I like receptor (RLR) signaling. This work focuses on the effect of TRBP on RIG-I signaling and IFN production. Our results indicate that TRBP acts as an inhibitor of RIG-I signaling in a PACT- and PKR-independent manner. This work has major implications on viral susceptibility, disease progression, and antiviral immunity as it demonstrates the regulatory interplay between two dsRNA binding proteins PACT and TRBP on RIG-I mediated IFN production. http://dx.doi.org/10.1016/j.cyto.2015.08.131

ID: 105 Activation of type I and III interferon response by mitochondrial and peroxisomal MAVS and inhibition by hepatitis C virus Silke Bender 1,2,*, Antje Reuter 2, Florian Eberle 1, Evelyne Einhorn 2, Marco Binder 2, Ralf Bartenschlager 1,2, 1 Department of Infectious Diseases, Molecular Virology, Heidelberg University, Germany, 2 Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany * Corresponding author at: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Germany. Sensing viruses by pattern recognition receptors (PRR) triggers the innate immune system of the host cell and activates immune signaling cascades like the RIG-I/IRF3 pathway. Mitochondrial antiviral-signaling protein (MAVS) is the crucial adaptor protein of this pathway localized on mitochondria, peroxisomes and mitochondria associated membranes (MAMs). Activation of MAVS leads to the production of type I and type III interferon (IFN) as well as interferon stimulated genes (ISGs). To refine the role of MAVS subcellular localization for the induction of type I and III IFN responses in hepatocytes, we generated cell lines with organelle-targeted MAVS and characterized innate immune response after viral infection. We established various functional and genetic knock-out cell systems reconstituted to express exclusively mitochondrial or peroxisomal MAVS. Infection with diverse RNA viruses mounted comparable levels of type I and III IFN expression irrespective of MAVS subcellular localization. To determine whether viral counteraction of MAVS is affected in these systems we employed infection of cells with the hepatitis C virus (HCV), a major causative agent of chronic liver diseases with a high propensity to establish persistence. This virus efficiently cleaves MAVS via a viral protease residing in nonstructural protein 3 (NS3). We found that both mitochondrial and peroxisomal MAVS were efficiently cleaved by NS3 and this cleavage was required to suppress activation of the IFN response. Taken together, our findings indicate comparable activation of the IFN response by peroxisomal and mitochondrial MAVS in hepatocytes and efficient counteraction of both MAVS species by the HCV NS3 protease.

http://dx.doi.org/10.1016/j.cyto.2015.08.132

ID: 106 IL-17A correlates with the nitric oxide pathway in immune host response during human cystic echinococcosis Dalila Mezioug *, Chafia Touil-Boukoffa, University of Sciences and Technology Houari Boumediene (U.S.T.H.B), Algeria * Corresponding author. Human cystic echinococcosis is a severe parasitic disease caused by the larval stage of Echinococcus granulosus. This parasitic infection usually manifests as unilocular cyst(s) mainly located in the liver and/or lungs or other viscera of intermediate host. It constitutes a serious public health problem in various parts of the world. The aim of this work was to study the involvement of IL-17A in host defense against E. granulosus infection. In this way, we investigate IL-17A, IL-6 and NO production in sera from Algerian patients with cystic echinococcosis and in supernatants culture of peripheral blood mononuclear cells (PBMCs) from the same patients stimulated by a major parasitic antigen. In the same way, the expression of STAT3and inducible NOS (NOS2) was measured in PBMCs of patients. We also studied NO modulation by IL-17A in PBMCs and monocytes cultures from patients in presence of E. granulosus protoscoleces (larval form of parasite). Analysis of cytokines and NO production revealed that the levels of IL-17A, IL-6

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and NO were elevated in all sera and PBMC culture supernatants from patients. Interestingly, our results show a significant positive correlation between NO and IL17A and IL-6 levels. We observed with interest that both STAT3 and NOS2 expression was upregulated in PBMCs. We noted with interest that co-cultures treatment with IL-17 caused an increased NO production and a decrease in the percentage of viable protoscoleces. Collectively, our results indicates that the IL-17A play a pivotal role in host defense against E. granulosus infection through the NO pathway. http://dx.doi.org/10.1016/j.cyto.2015.08.133

ID: 107 Inhibition of type I IFN induction by Sandfly fever Sicilian virus virulence factor NSs Jennifer Wuerth 1,*, Matthias Habjan 2, Andreas Pichlmair 2, Giulio Superti-Furga 3, Friedemann Weber 1,4, 1 Philipps-University Marburg, Germany, 2 Max Planck Institute of Biochemistry, Martinsried, Germany, 3 Center for Molecular Medicine, Vienna, Austria, 4 Justus-Liebig University, Gießen, Germany * Corresponding author. Phleboviruses are a group of emerging viruses with a wide spectrum of virulence: For example, Rift Valley Fever virus (RVFV) is highly pathogenic, whereas Sandfly fever Sicilian virus (SFSV) causes intermediate pathogenicity. The major virulence factor is the non-structural protein NSs, a suppressor of the type I interferon (IFN) system. While the NSs protein of highly pathogenic RVFV inhibits global host cell transcription via sequestration and degradation of the TFIIH subunits p44 and p62, respectively, the mode of action of the NSs protein of intermediately pathogenic SFSV has remained elusive. We therefore aimed to characterize the IFN-inhibitory function employed by the NSs of SFSV. Using mass spectrometry, our group has identified multiple candidate host interactors, among them interferon regulatory factor 3 (IRF3). Indeed, co-immunoprecipitation confirmed the interaction of SFSV NSs with IRF3, but not with other IRF family members like IRF2, IRF5 and IRF9. Furthermore, SFSV NSs specifically abrogated IRF3 activation and thus IFN-b promoter activity, but not general host cell transcription. Hence, SFSV encodes a NSs protein that efficiently inhibits the induction of type I IFN. Different to the highly pathogenic RVFV, which induces a general host transcription shutoff, however, SFSV specifically targets IRF3-dependent IFN promoter activation. Although RVFV and SFSV are highly related, their NSs proteins display remarkably diverse strategies of counteracting the type I IFN system. Possibly, the quality of NSshost factor interactions correlates with the virulence levels of phleboviruses. http://dx.doi.org/10.1016/j.cyto.2015.08.134

ID: 108 Spatial insights in the interaction between the RIG-I like receptor pathway and viral proteins Maria Teresa Sánchez-Aparicio 1,2, Juan Ayllon 1,2, Adolfo García-Sastre 1,2,3, Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 2 Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 3 Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA 1

The innate immune response relies on a set of Pathogen Recognition Receptors (PRRs) that sensor pathogen patterns (PAMPs). RIG-I is a cytosolic PRR that detects 5’-triphosphate double-stranded RNAs produced during infection. Once activated, the pathway leads to the induction of type I IFN and proinflammatory cytokines, leading to a cellular antiviral state. Upon recognition of the RNA, RIG-I hydrolyzes ATP and changes its conformation to an active state. The CARD domains are then exposed and become K63-linked polyubiquitinated by E3-ligases, such as TRIM25. The activation of this pathway is complex and well characterized, but most of the spatio-temporal events, and the subcellular localization where the essential proteins interact, are still under interrogation. Through different techniques, we analyzed how these proteins form complexes that are distributed and reorganized spatially within the cell in order to create an efficient antiviral state. RIG-I is the main sensor for recognition of viruses such as Paramyxoviruses, Flaviviruses, Rhabdoviruses and Orthomyxoviruses. Many of them have developed numerous and different strategies to overcome the activation of the RLR pathway. We will discuss and show new insights on how, where, and when, viral proteins can counteract the activation of the RLR pathway. NS1 of Influenza A virus, or NS3/4A of Hepatitis C Virus, as an example, are IFN antagonistic viral proteins that interact with specific complexes in very well defined areas in the host cell in order to inhibit the antiviral state in an infected cell. http://dx.doi.org/10.1016/j.cyto.2015.08.135