IFN Regulatory Factor-1 Bypasses IFN-Mediated Antiviral Effects through Viperin Gene Induction This information is current as Anja Stirnweiss, Antje Ksienzyk, Katjana Klages, Ulfert of October 1, 2021. Rand, Martina Grashoff, Hansjörg Hauser and Andrea Kröger J Immunol 2010; 184:5179-5185; Prepublished online 22 March 2010; doi: 10.4049/jimmunol.0902264 http://www.jimmunol.org/content/184/9/5179 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2010/03/22/jimmunol.090226 Material 4.DC1 http://www.jimmunol.org/ References This article cites 35 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/184/9/5179.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on October 1, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology IFN Regulatory Factor-1 Bypasses IFN-Mediated Antiviral Effects through Viperin Gene Induction Anja Stirnweiss,1 Antje Ksienzyk, Katjana Klages,2 Ulfert Rand, Martina Grashoff, Hansjo¨rg Hauser, and Andrea Kro¨ger Viperin is an antiviral protein whose expression is highly upregulated during viral infections via IFN-dependent and/or IFN- independent pathways. We examined the molecular alterations induced by the transcriptional activator IFN regulatory factor (IRF)-1 and found viperin to be among the group of IRF-1 regulated genes. From these data, it was not possible to distinguish genes that are primary targets of IRF-1 and those that are targets of IRF-1–induced proteins, like IFN-b. In this study, we show that IRF-1 directly binds to the murine viperin promoter to the two proximal IRF elements and thereby induces viperin expression. Infection studies with embryonal fibroblasts from different gene knock-out mice demonstrate that IRF-1 is essential, whereas the type I IFN system is dispensable for vesicular stomatitis virus induced viperin gene transcription. Further, IRF-1, but Downloaded from not IFN type I, mediates the induction of viperin transcription after IFN-g treatment. In contrast, IRF-1 is not required for IFN- independent viperin induction by Newcastle disease virus infection and by infection with a vesicular stomatitis virus mutant that is unable to block IFN expression and secretion. We conclude that the IRF-1 mediated type I IFN independent mechanism of enhanced viperin expression provides a redundant mechanism to protect cells from viral infections. This mechanism becomes important when viruses evade innate immunity by antagonizing the induction and function of the IFN system. The Journal of Immunology, 2010, 184: 5179–5185. http://www.jimmunol.org/ he early innate immune system defends against infections and myxovirus resistance gene 1 are nevertheless able to mount by eliciting a nonspecific response after the recognition of a substantial, residual antiviral response (2). For the majority of T various pathogen-associated molecular patterns. TLRs and ISG-encoded proteins, the exact molecular mechanisms un- members of the retinoic acid inducible gene family recognize derlying their antiviral actions remain unknown. foreign nucleic acids within endosomal and cytoplasmic com- Viperin (virus inhibitory protein, endoplasmatic reticulum- partments, initiating a signaling cascade that involves the induction associated, IFN-inducible), an highly conserved antiviral ISG, was of type I IFN through the transcription factors IFN regulatory factor initially identified as a human CMV (HCMV)-inducible protein in by guest on October 1, 2021 (IRF) 3 and NFkB (1). IFNs represent the first line of defense primary skin cultures (3). Its expression is highly upregulated by against viral infection and mediate their antiviral activities by the type I, II and III IFNs (4–6), microbial products like LPS (7–9), induction of IFN-stimulated genes (ISGs). These IFN-stimulated and by infection with a broad range of viruses (4, 8–12), sug- mediators interfere with many steps of the viral replication process gesting an important role in the innate immune response. Fur- through mechanisms that are extremely complex and involve thermore, data from viperin-deficient mice indicate that viperin is multiple overlapping or related pathways. For instance, mice that required for optimal Th2 response and thus is involved in adaptive are triply deficient for RNase L, RNA-dependent protein kinase R, immune responses as well (13). Viperin interferes with the prop- agation of a broad spectrum of viruses, for example, HCMV, HIV, hepatitis-C virus (HCV), alfavirus, and influenza A virus (10, 11, Department of Gene Regulation and Differentiation, Helmholtz Centre for Infection 14–16). In HCMV infected cells, the expression of viperin inhibits Research, Braunschweig, Germany the expression of several structural proteins that are critical for 1Current address: Institute of Molecular and Clinical Immunology, Otto-von-Guericke viral assembly and maturation (10). In influenza A virus-infected University of Magdeburg, Magdeburg, Germany. cells, viperin prevents virus release from the plasma membranes 2 Current address: Department of Experimental Immunology, Helmholtz Centre for by influencing the farnesyl diphospate synthetase, which is re- Infection Research, Braunschweig, Germany. sponsible for the organization of lipid rafts (11). Recently, viperin Received for publication July 15, 2009. Accepted for publication February 22, 2010. had been shown to reduce the secretion of soluble, but not This work was supported by the German Research Foundation (SFB 566, B7), the membrane-bound proteins and to alter the endoplasmatic re- German Ministry of Research and Education (FORSYS-Partner), Deutsche Kreb- shilfe (10-2136), and Wilhelm Sander Stiftung. ticulum membrane structure (17). How this contributes to the Address correspondence and reprint requests to Dr. Andrea Kro¨ger, Department of antiviral capability of viperin remains unclear. Gene Regulation and Differentiation, Helmholtz Centre for Infection Research, In- Given the critical role of innate immune response in survival from hoffenstraße 7, D-38124 Braunschweig, Gemany. E-mail address: andrea.kroeger@ infection, it is not surprising that a number of molecules involved in helmholtz-hzi.de virus recognition and IFN signaling, for example,IRF-3 and IFN itself The online version of this article contains supplemental material. are affected by viral proteins (18). Cells infected with Japanese en- Abbreviations used in this paper: CHX, cycloheximide; HCMV, human cytomegalo- virus; HCV, hepatitis-C virus; IRF, IFN regulatory factor; IRF-E, IRF element; ISG, cephalitis virus fail to clear the virus infection, because through a yet IFN stimulated gene; ISGF3, ISG factor 3; ISRE, IFN stimuated regulatory element; unknown mechanism thevirus evades the antiviral effect of viperin by MEF, mouse embryonic fibroblasts; MOI, multiplicity of infection; NDV, Newcastle degrading the protein (12). The existence of these viral mechanisms disease virus; VSV, vesicular stomatitis virus; wt, wild-type. forced evolution to develop redundant pathways for detection and Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 inhibition of viral infections. The findings of several groups revealed www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902264 5180 IFN INDEPENDENT, IRF-1 DEPENDENT VIPERIN INDUCTION that the activation of viperin expression can result through IFN- Luciferase Kit (Promega, Madison, WI). Signals were normalized to the independent mechanisms, depending on the infecting virus (8, 9, 12). Renilla luciferase activity. All experiments were performed at least twice In this study, we describe the transcription factor IRF-1 as an essential with triplicates before calculating mean values and standard deviations. mediator of viperin induction, acting through an IFN-independent Electro mobility shift assay pathway in vesicular stomatitis virus (VSV) infected cells. We also Murine IRF-1 and firefly luciferase were in vitro translated using the TNT investigated the molecular basis for the IRF-1 induced promoter ac- T7 Quick Coupled Transcription/Translation System (Promega), according tivation. Furthermore, we show that an enhanced viperin expression, to the manufacturer’s protocol. pMT7-IRF-1 and luciferase T7 control DNA comparable to that in VSV infected cells, results in a reduction of served as templates. EMSA experiments were performed with 5 mlinvitro VSV replication even in the absence of functional IFN signaling. translated protein mix in the absence or presence of 1 mganti-IRF-1(M-20; Santa Cruz Biotechnology, Santa Cruz, CA), as described in Klar and Bode (22). The used oligonucleotides were shown in the Supplemental Table II. Materials and Methods Cell culture and treatments IFN assay IFNAR2/2, IRF-12/2, and STAT12/2 mouse embryonic fibroblasts