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      Structures of Arenaviral Nucleoproteins with Triphosphate dsRNA Reveal a Unique Mechanism of Immune Suppression*

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          Abstract

          Background: Arenaviral nucleoproteins play a critical role in innate immune suppression.

          Results: Structures of Lassa nucleoprotein in complex with triphosphate dsRNA and Tacaribe virus nucleoprotein have been determined.

          Conclusion: Both Lassa and Tacaribe nucleoproteins can strongly inhibit IFN-β production by degrading immune-stimulatory dsRNA.

          Significance: A unique immune suppression mode of arenaviral nucleoproteins has been revealed.

          Abstract

          A hallmark of severe Lassa fever is the generalized immune suppression, the mechanism of which is poorly understood. Lassa virus (LASV) nucleoprotein (NP) is the only known 3′-5′ exoribonuclease that can suppress type I interferon (IFN) production possibly by degrading immune-stimulatory RNAs. How this unique enzymatic activity of LASV NP recognizes and processes RNA substrates is unknown. We provide an atomic view of a catalytically active exoribonuclease domain of LASV NP (LASV NP-C) in the process of degrading a 5′ triphosphate double-stranded (ds) RNA substrate, a typical pathogen-associated molecular pattern molecule, to induce type I IFN production. Additionally, we provide for the first time a high-resolution crystal structure of an active exoribonuclease domain of Tacaribe arenavirus (TCRV) NP. Coupled with the in vitro enzymatic and cell-based interferon suppression assays, these structural analyses strongly support a unified model of an exoribonuclease-dependent IFN suppression mechanism shared by all known arenaviruses. New knowledge learned from these studies should aid the development of therapeutics against pathogenic arenaviruses that can infect hundreds of thousands of individuals and kill thousands annually.

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          Activation of MDA5 requires higher-order RNA structures generated during virus infection.

          Recognition of virus presence via RIG-I (retinoic acid inducible gene I) and/or MDA5 (melanoma differentiation-associated protein 5) initiates a signaling cascade that culminates in transcription of innate response genes such as those encoding the alpha/beta interferon (IFN-alpha/beta) cytokines. It is generally assumed that MDA5 is activated by long molecules of double-stranded RNA (dsRNA) produced by annealing of complementary RNAs generated during viral infection. Here, we used an antibody to dsRNA to show that the presence of immunoreactivity in virus-infected cells does indeed correlate with the ability of RNA extracted from these cells to activate MDA5. Furthermore, RNA from cells infected with encephalomyocarditis virus or with vaccinia virus and precipitated with the anti-dsRNA antibody can bind to MDA5 and induce MDA5-dependent IFN-alpha/beta production upon transfection into indicator cells. However, a prominent band of dsRNA apparent in cells infected with either virus does not stimulate IFN-alpha/beta production. Instead, stimulatory activity resides in higher-order structured RNA that contains single-stranded RNA and dsRNA. These results suggest that MDA5 activation requires an RNA web rather than simply long molecules of dsRNA.
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            5'-triphosphate RNA requires base-paired structures to activate antiviral signaling via RIG-I.

            The ATPase retinoid acid-inducible gene (RIG)-I senses viral RNA in the cytoplasm of infected cells and subsequently activates cellular antiviral defense mechanisms. RIG-I recognizes molecular structures that discriminate viral from host RNA. Here, we show that RIG-I ligands require base-paired structures in conjunction with a free 5'-triphosphate to trigger antiviral signaling. Hitherto unavailable chemically synthesized 5'-triphosphate RNA ligands do not trigger RIG-I-dependent IFN production in cells, and they are unable to trigger the ATPase activity of RIG-I without a base-paired stretch. Consistently, immunostimulatory RNA from cells infected with a virus recognized by RIG-I is sensitive to double-strand, but not single-strand, specific RNases. In vitro, base-paired stretches and the 5'-triphosphate bind to distinct sites of RIG-I and synergize to trigger the induction of signaling competent RIG-I multimers. Strengthening our model of a bipartite molecular pattern for RIG-I activation, we show that the activity of supposedly "single-stranded" 5'-triphosphate RNAs generated by in vitro transcription depends on extended and base-paired by-products inadvertently, but commonly, produced by this method. Together, our findings accurately define a minimal molecular pattern sufficient to activate RIG-I that can be found in viral genomes or transcripts.
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              Recognition of viruses by cytoplasmic sensors.

              The immune response to virus infection is initiated when pathogen recognition receptors (PRRs) of the host cell recognize specific nonself-motifs within viral products (known as a pathogen-associated molecular pattern or PAMP) to trigger intracellular signaling events that induce innate immunity, the front line of defense against microbial infection. The replication program of all viruses includes a cytosolic phase of genome amplification and/or mRNA metabolism and viral protein expression. Cytosolic recognition of viral infection by specific PRRs takes advantage of the dependence of viruses on the cytosolic component of their replication programs. Such PRR-PAMP interactions lead to PRR-dependent nonself-recognition and the downstream induction of type I interferons and proinflammatory cytokines. These factors serve to induce innate immune programs and drive the maturation of adaptive immunity and inflammation for the control of infection. Recent studies have focused on identifying the particular viral ligands recognized as nonself by cytosolic PRRs, and on defining the nature of the PRRs and their signaling pathways involved in immunity. The RIG-I-like receptors, RIG-I and MDA5, have been defined as essential PRRs for host detection of a variety of RNA viruses. Novel PRRs and their signaling pathways involved in detecting DNA viruses through nonself-recognition of viral DNA are also being elucidated. Moreover, studies to identify the PRRs and signaling factors of the host cell that mediate inflammatory signaling through inflammasome activation following virus infection are currently underway and have already revealed specific NOD-like receptors (NLRs) as inflammatory triggers. This review summarizes recent progress and current areas of focus in pathogen recognition and immune triggering by cytosolic PRRs. Copyright 2009 Elsevier Ltd. All rights reserved.
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                7 June 2013
                24 April 2013
                24 April 2013
                : 288
                : 23
                : 16949-16959
                Affiliations
                From the []Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom,
                the [§ ]Department of Veterinary and Biomedical Sciences, University of Minnesota, Twin Cities, St. Paul, Minnesota 55108,
                the []Laboratory of Department of Surgery, the First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong 510080, China, and
                the []Biomedical Sciences Research Complex, University of St. Andrews, St. Andrews KY16 9ST, United Kingdom
                Author notes
                [2 ] To whom correspondence may be addressed. Tel.: 612-625-3358; E-mail: hly@ 123456umn.edu .
                [3 ] To whom correspondence may be addressed. Tel.: 612-625-3376; E-mail: liangy@ 123456umn.edu .
                [4 ] To whom correspondence may be addressed. Tel.: 01603-591739; E-mail: c.dong@ 123456uea.ac.uk .
                [1]

                Both authors contributed equally to this work.

                Article
                M112.420521
                10.1074/jbc.M112.420521
                3675627
                23615902
                6ffa8c31-7846-4eca-9757-496177032764
                © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version full access.

                Creative Commons Attribution Unported License applies to Author Choice Articles

                History
                : 11 February 2013
                : 15 April 2013
                Funding
                Funded by: National Institutes of Health
                Award ID: R01AI083409
                Award ID: R01AI093580
                Award ID: R56AI091805
                Categories
                Molecular Bases of Disease

                Biochemistry
                immunosuppression,nucleic acid enzymology,protein structure,protein-nucleic acid interaction,viral protein,3′-5′ exoribonuclease,lassa fever virus,tacaribe virus,type i interferons,immune evasion

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